Immunological Targeting of Pathological Tau Proteins

ABSTRACT

The present invention relates to methods and compositions for treating, preventing, and diagnosing Alzheimer&#39;s Disease or other tauopathies in a subject by administering an immunogenic tau peptide or an antibody recognizing the immunogenic tau epitope under conditions effective to treat, prevent, or diagnose Alzheimer&#39;s Disease or other tauopathies. Also disclosed are methods of promoting clearance of aggregates from the brain of the subject and of slowing progression of tau-pathology related behavioral phenotype in a subject.

This application claims priority to U.S. Non-Provisional patentapplication Ser. No. 12/813,297, filed Jun. 10, 2010 (pending) and U.S.Provisional Patent Application Ser. No. 61/185,895, filed Jun. 10, 2009(expired), each of which applications is hereby incorporated byreference in its entirety.

The subject matter of this application was made with support from theUnited States Government under the National Institutes of Health, GrantNo. AG032611. The U.S. Government has certain rights.

FIELD OF THE INVENTION

The present invention is directed to immunological methods andcompositions for preventing, treating, and diagnosing Alzheimer'sdisease and related tauopathies, and inhibiting the accumulation of tauneurofibrillary tangles and/or their pathological tau precursors in asubject.

BACKGROUND OF THE INVENTION

An emerging treatment for Alzheimer's disease (AD) is immunotherapy toclear amyloid-β (Aβ). Another important target in AD and frontotemporaldementia is the neurofibrillary tangles and/or their pathological tauprotein conformers, whose presence correlates well with the degree ofdementia (Terry R., “Neuropathological Changes in Alzheimer Disease,”Prog Brain Res. 101:383-390 (1994); Goedert M., “Tau Protein andNeurodegeneration,” Semin Cell Dev Biol. 15:45-49 (2004)). The objectiveof immunotherapy for tau pathology is that anti-tau antibodies can cleartau aggregates that may affect neuronal viability. Other components ofthe immune system may play a role as well in the clearance. Tau is asoluble protein that promotes tubulin assembly, microtubule stability,and cytoskeletal integrity. Although tau pathology is likely to occurfollowing Aβ aggregation based on Down syndrome studies, analyses of ADbrains and mouse models indicate that these pathologies are likely to besynergistic (Sigurdsson et al., “Local and Distant 30 HistopathologicalEffects of Unilateral Amyloid-beta 25-35 Injections into the Amygdala ofYoung F344 Rats,” Neurobiol Aging 17:893-901 (1996); Sigurdsson et al.,“Bilateral Injections of Amyloid-β 25-35 into the Amygdala of YoungFischer Rats: Behavioral, Neurochemical, and Time DependentHistopathological Effects,” Neurobiol Aging 18:591-608 (1997); Lewis etal., “Enhanced Neurofibrillary Degeneration in Transgenic MiceExpressing Mutant Tau and APP,” Sience 293(5534):1487-91 (2001): Gotz etal., “Formation of Neurofibrillary Tangles in P301L Tau Transgenic MiceInduced by A-beta 42 Fibrils,” Science 293:1491-1495 (2001); Delacourteet al., “Nonoverlapping but Synergetic Tau and APP Pathologies inSporadic Alzheimer's Disease,” Neurology. 59:398-407 (2002); Oddo etal., “Abeta Immunotherapy Leads to Clearance of Early, But Not Late,Hyperphosphorylated Tau Aggregates via the Proteasome,” Neuron43:321-332 (2004); Ribe et al., “Accelerated Amyloid Deposition,Neurofibrillary Degeneration and Neuronal Loss in Double Mutant APP/TauTransgenic Mice,” Neurobiol Dis. (2005)). Hence, targeting bothpathologies may substantially increase treatment efficacy. To date, notau mutations have been observed in AD, however, in frontotemporaldementia, mutations in the tau protein on chromosome 17 (FTDP-17) are acausative factor in the disease, which further supports tau-basedtherapeutic approaches (Poorkaj et al., “Tau is a Candidate Gene forChromosome 17 Frontotemporal Dementia,” Ann Neurol. 43:815-825 (1998);Spillantini et al., “Frontotemporal Dementia and Parkinsonism Linked toChromosome 17: A New Group of Tauopathies,” Brain Pathol. 8:387-402(1998)). Transgenic mice expressing these mutations have modeled manyaspects of the disease and are valuable tools to study the pathogenesisof tau-pathology related neurodegeneration and to assess potentialtherapies. One of these models, the P301L mouse model (Lewis et al.,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat. Genet. 25:402-405(2000)), recapitulates many of the features of frontotemporal dementiaalthough the CNS distribution of the tau aggregates results primarily insensorimotor abnormalities which complicates cognitive assessment.Homozygous lines of this mouse model have an early onset of CNSpathology and associated functional impairments which make them idealfor the initial assessment of the feasibility of immunotherapy,targeting pathological tau conformers.

Other tau-related therapeutic approaches include: (1) drugs that inhibitthe kinases or activate the phosphatases that affect the state of tauphosphorylation (Iqbal et al., “Inhibition of NeurofibrillaryDegeneration: A Promising Approach to Alzheimer's Disease and OtherTauopathies,” Curr Drug Targets 5:495-502 (2004); Noble et al.,Inhibition of Glycogen Synthase Kinase-3 by Lithium Correlates withReduced Tauopathy and Degeneration In Vivo,” Proc Natl Acad Sci USA102:6990-6995 (2005)); (2) microtubule stabilizing drugs (Michaelis etal. {beta}-Amyloid-Induced Neurodegeneration and Protection byStructurally Diverse Microtubule-Stabilizing Agents,” J Pharmacol ExpTher. 312:659-668 (2005); Zhang et al., “Microtubule-Binding DrugsOffset Tau Sequestration by Stabilizing Microtubules and Reversing FastAxonal Transport Deficits in a Tauopathy Model,” Proc Natl Acad Sci USA102:227-231 (2005)); (3) compounds that interfere with tau aggregation(Pickhardt et al., “Anthraquinones Inhibit Tau Aggregation and DissolveAlzheimer's Paired Helical Filaments In Vitro and in Cells,” J Biol.Chem. 280:3628-3635 (2005)); and (4) drugs that promote heat shockprotein mediated clearance of tau (Dickey et al., “Development of a HighThroughput Drug Screening Assay for the Detection of Changes in TauLevels—Proof of Concept with HSP90 Inhibitors,” Curr Alzheimer Res.2:231-238 (2005)). While all these approaches are certainly worthpursuing, target specificity and toxicity are of a concern, whichemphasizes the importance of concurrently developing other types oftau-targeting treatments, such as immunotherapy.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method ofpreventing or treating Alzheimer's disease or other tauopathy in asubject. This method involves administering, to the subject, any one ormore immunogenic tau peptides having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2-75, or one or more antibodiesrecognizing an immunogenic tau epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-75 and 101-103 underconditions effective to treat or prevent Alzheimer's disease or othertauopathy in the subject.

Another aspect of the present invention is directed to a method ofpromoting clearance of tau aggregates from the brain of a subject. Thismethod involves administering, to the subject, any one or moreimmunogenic tau peptides having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-75, or one or more antibodiesrecognizing an immunogenic tau epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-75 and 101-103 underconditions effective to promote clearance of the tau aggregates from thebrain of the subject.

A third aspect of the present invention is directed to a method ofslowing progression of a tau-pathology related behavioral phenotype in asubject. This method involves administering, to the subject, any one ormore immunogenic tau peptides having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2-75, or one or more antibodiesrecognizing an immunogenic tau epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-75 and 101-103 underconditions effective to slow the progression of the tau-pathologyrelated behavioral phenotype in the subject.

A fourth aspect of the present invention is directed to an isolated taupeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2-75 and 101-103. The immunogenic tau peptideis effective in preventing and treating Alzheimer's disease or othertauopathy in a subject, promoting the clearance of aggregates from thebrain of a subject, and slowing the progression of a tau-pathologyrelated behavioral phenotype in a subject.

Neurofibrillary tangles and their pathological tau protein conformersare important targets for preventing and treating Alzheimer's diseaseand other tau-related neurodegenerative diseases. However, a strategyfor targeting and clearing neurofibrillary tangles and/or pathologicaltau conformers that has high target specificity and minimal to notoxicity is lacking. The immunogenic tau peptides and antibodiesdescribed herein were designed to overcome this deficiency. Because theimmunogenic tau peptides of the present invention mimic narrowphospho-epitopes of the pathological tau, and the tau antibodiesrecognize these same narrow phospho-epitopes, enhanced specificity andsafety are achieved. This scenario also applies to the antibodiesdescribed herein that are generated against the free N- or C-terminus ofpathological tau fragments. Accordingly, using the immunotherapeuticapproaches described herein, a robust immune response against thepathological tau protein can be generated with minimal risk of producingan adverse immune response towards the normal tau protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict the immune response in the JNPL3 P301L tangle mousemodel to tau immunogenic peptides of the present invention. Mice of 2-3months of age received the first two immunizations two weeks apart andthen monthly thereafter. To assess antibody response, the mice were bledprior to the first immunization, periodically thereafter one week aftervaccine administration, and when the mice were killed for tissueharvesting at 8-9 months of age. The IgG and IgM antibody response shownin FIGS. 1A and 1B was measured one week after the 6^(th) immunization(T3) and again at 8-9 months of age, which was at the time of sacrifice(Tf=Tfinal). FIG. 1A shows a robust IgG and IgM immune response in JNPL3P301L mice immunized with Tau210-216[P-Thr₂₁₂-Ser₂₁₄] (SEQ ID NO: 2)linked to tetanus toxin helper T-cell epitope (TT947-967) via GPSLlinker. FIG. 1B shows that a strong antibody response is generatedagainst the tetanus toxin epitope as assessed by IgG and IgM binding toan unrelated tau epitope Tau260-264[P-Ser₂₆₂] linked via GPSL toTT947-967. ELISA plates were coated with 0.5 μg peptide per well andplasma was diluted 1:200.

FIGS. 2A-2C show that JNPL3 P301L tangle mice immunized withTau260-264[P-Ser₂₆₂] (SEQ ID NO: 3) (also referred to the T299 peptide)linked to tetanus toxin helper T-cell epitope (TT947-967) via a GPSLlinker generate a robust IgG response against the immunogen. FIG. 2Ashows the IgG antibody response in mice following immunization with theTau260-264[P-Ser₂₆₂] peptide. As above, the mice received the first twoimmunizations two weeks apart and then monthly thereafter from 2-3months of age until 8-9 months of age. FIG. 2B shows that a good portionof the antibody response is generated against the tetanus toxin epitopeas assessed by IgG binding to an unrelated tau epitopeTau210-216[P-Thr₂₁₂-Ser₂₁₄] linked via GPSL to TT947-967. FIG. 2C showsthat a good portion of the antibody response is generated against thetau epitope as assessed by IgG binding to a larger tau epitopeTau240-270[P-Ser₂₆₂] that contains the Tau260-264[P-Ser₂₆₂] region.ELISA plates were coated with 0.5 μg peptide per well and plasma wasdiluted 1:200. T0-Tfinal: Bleed prior to vaccination (T0), one weekafter third -(T1), sixth -(T2), seventh (T3) immunization, and at tissueharvesting (Tf).

FIG. 3 shows the robust antibody (IgG) response generated in JNPL3 P301Ltangle model mice immunized in with Tau229-237[P-Thr₂₃₁-Ser₂₃₅] (SEQ IDNO: 4) linked to tetanus toxin helper T-cell epitope (TT947-967]. Themice were immunized from 2-3 months of age, two weeks apart, then amonth later, and bled (T1) one week after the third immunization. ELISAplates were coated with 0.5 μg peptide per well and plasma was diluted1:200.

FIG. 4 shows the robust antibody (IgG) response generated in JNPL3 P301Ltangle model mice immunized with the pseudophosphorylated immunogen,Tau379-408[Asp_(396, 404)] (SEQ ID NO: 57) in alum adjuvant.Importantly, these antibodies recognize the phospho-epitope,Tau379-408[P-Ser_(396, 404)], to a similar degree. The mice wereimmunized from 2-3 months of age, two weeks apart for the first twoimmunizations, and monthly thereafter. The mice were bled (Tf=Tfinal) atthe time of tissue harvesting at 8-9 months of age. ELISA plates werecoated with 0.5 μg peptide per well and plasma was diluted 1:200.

FIG. 5A-5B show the reduction of pathological tau observed in the brainstem (FIG. 5A) and dentate gyrus (FIG. 5B) of the tangle mouse modelfollowing tau immunotherapy. Homozygous JNPL3 tau P301L mice wereimmunized with T299 (Tau260-264[P-Ser₂₆₂] (SEQ ID NO: 3)) linked to atetanus toxin helper T-cell epitope (TT1947-967) via a GPSL linkersequence. Pathological tau in both the brain stem and dentate gyrus wereassessed by PHF1 antibody immunostaining. PHF1 is a monoclonal antibodyrecognizing tau that is phosphorylated on serine amino acids 404 and 396on the C-terminal (Greenberg et al., “Hydrofluoric Acid-Treated Tau PHFProteins Display the Same Biochemical Properties as Normal Tau,” J BiolChem 267:564-569 (1992), which is hereby incorporated by reference inits entirety). A significant reduction of pathological tau staining wasobserved in both the brain stem and dentate gyrus of animals activelyimmunized with the T299 peptide compared to control animals receivingadjuvant only.

FIG. 6 shows that immunization of htau/PS1 mice with the phosphorylatedTau379-408[P-Ser_(396,404)] (SEQ ID NO: 82) reduces the amount of tauaggregates by 56% in the pyriform cortex. Significant difference wasobserved between the immunized and control groups (one-way ANOVA,p<0.01). Post hoc analysis also showed that immunized htau/PS1 micediffered from their htau/PS1 controls (p<0.01). ** p<0.01.

FIGS. 7A-7B show that tau immunotherapy prevents functional impairmentin a tangle mouse model. Homozygous JNPL3 P301L mice were immunized withthe phosphorylated immunogenic Tau 299 peptide (Tau260-264[P-Ser₂₆₂](SEQ ID NO:3)) linked to a tetanus toxin helper T-cell epitope(TT947-967) via a GPSL linker sequence. Control animals receivedadjuvant alone. Administration of the Tau260-264[P-Ser₂₆₂] peptidevaccine prevented functional impairments assessed using the traversebeam at 8 months of age as indicated by the fewer number of footslipsrecorded for the immunized animals compared to the control animals (FIG.7A). Likewise, administration of the Tau260-264[P-Ser₂₆₂] peptidevaccine prevented functional impairments assessed by the rotarod test,both at 5-6 months of age and at 8-9 months of age (FIG. 7B).

FIGS. 8A-8B show that immunization of htau/PS1 mice with thephosphorylated Tau379-408[P-Ser_(396,404)] (SEQ ID NO:82) improvesperformance in the radial arm maze (FIG. 8A) and the object recognitiontest (FIG. 8B). A significant difference was observed between theimmunized and control groups in the radial arm maze (two-way ANOVArepeated measures, p<0.0001) as shown in FIG. 8A. Neuman-Keuls post-hoctest revealed that the immunized htau/PS1 mice performed better (i.e.,committed less errors) than the control htau/PS1 mice on all the days(p<0.01-0.001). A significant difference was also observed between thegroups in the object recognition test (one-way ANOVA, p=0.005) (FIG.8B). Neuman-Keuls post-hoc test revealed that the immunized htau/PS1mice had better short-term memory than identical control mice (p<0.01).It has been well established that cognitively normal mice spend about70% of their time with the new object compared to the old object. **p<0.01.

FIGS. 9A-9C show that immunization of htau/PS1 mice with thephosphorylated Tau379-408[P-Ser_(396,404)] (SEQ ID NO:82) improvesperformance in the closed field symmetrical maze. Significantdifferences were observed between the immunized and control groups withrespect to the number of errors committed in each of mazes 9A-9C(one-way ANOVA, Maze A: p<0.001. Maze B: p<0.0001, Maze C: p<0.01).Post-hoc analysis revealed that the treated htau/PS1 group performedbetter than their identical control mice (htau/PS1 controls) (Maze A:p<0.01, Mazes B, C: p<0.001). Post-hoc analysis also revealedsignificant differences between some of the other groups depending onthe maze but those differences are less relevant and are therefore notdetailed here. The three mazes were of increasing complexity asindicated by the number of errors (note that the Y axis scale differs).** p<0.01, *** p<0.001.

FIGS. 10A-10F are graphs depicting the levels of soluble and insolubletau (total tau and pathological tau) detected by western blot analysisin htau/PS1 mice immunized with phosphorylatedTau379-408[P-Ser_(396,404)] (SEQ ID NO:82) and corresponding controls.Tau immunotherapy reduces pathological tau compared to total tau by35-43% (FIGS. 10C and 10D). The immunotherapy did not affect total taulevels as assessed with B19 antibody (FIGS. 10A and 10B) which isimportant for the safety of this approach. Compared to htau/PS controls,PHF1 soluble tau was significantly reduced (p<0.001) and the soluble tauratio (PHF1/total tau) was reduced by 35% (p<0.05) (FIG. 10E). A strongtrend for reduction in PHF1 insoluble tau was observed as well (p=0.06),and the insoluble tau ratio (PHF1/total tau) was reduced by 43% (p=0.08)(FIG. 10F). * p<0.05. ***r p<0.001

FIGS. 11A-11C demonstrate that passive immunotherapy targeting thephosphorylated tau 396 and 404 epitopes prevents functional decline andreduces tau pathology in P301L tangle mice. FIG. 11A is a graph showinga significant difference in the number of footslips taken on thetraverse beam by IgG injected control and PHF1 immunized P301L mice,with control animals having more footslips when crossing the beam(trials combined, p=0.03). FIG. 11B is a graph showing the percentage oftau immunostaining in the dentate gyrus of immunized and control P301Lmice. PHF1 immunized P301L mice had 58% less PHF1 stained tau pathologyin the dentate gyrus than controls (p=0.02). As shown in FIG. 11C, theamount of PHF-1 antibodies (μg/μL) in plasma decreased four-fold in twoweeks. No detectable antibodies were observed in controls, whereas thelevels in immunized animals decreased over time. These are the averagevalues for the immunized mice. T0: prior to first immunization, T1: 24 hafter the 12th injection, T2: 7 days after the 13th and last injection,T3: 14 days after last injection. The ELISA plates were coated withTau379-408[P-Ser_(396,404)]

FIGS. 12A-2B are graphs showing the inverse correlation between plasmalevels of PHF1 antibodies and tau pathology. Significant correlation wasobserved in the brain stem (FIG. 12A; p<0.01), and a strong trend forcorrelation in the motor cortex (FIG. 12B: p=0.06).

FIGS. 13A-13B are graphs depicting the generation of monoclonalantibodies against the immunogenic tau peptide comprising amino acids386-408 (SEQ ID NO: 13) containing phosphorylated serine epitopes atamino acid positions 396 and 404. As shown in FIG. 13A, a very strongtiter was generated against the tau portion of the immunogenTau-386-408[P-Ser_(396,404)] (red) as detected by serial dilutions ofplasma. The plasma antibodies preferably recognized the phospho-Ser404epitope (blue) and the non-phospho epitope (white). The phospho-Ser396epitope (green) was recognized to a lesser degree. Numerous stronglypositive clones were detected (>50). Of those, 8 phospho-specific cloneswere selected for a first subcloning (FIG. 13B). All appeared stable andthree were selected for second subcloning (all IgG1). Of the clones thatdid not specifically recognize a phosphorylated-epitope, six wereselected for first subcloning. All appeared stable and three wereselected for second subcloning (IgG1, IgG2a and LgM).

FIGS. 14A-14B are graphs showing epitope binding of stablephospho-specific (FIG. 14A) and non-phospho-specific (FIG. 14B)Tau-386-408[P-Ser_(396,404)] antibody clones after third subcloning byELISA. Of the phospho-specific monoclonal antibodies selected forfurther subcloning, four out of six retained their specificity for thephospho-Ser₄₀₄ epitope (see clones 1F12C2, 1F12G6, 4E6E3, and 4E6G7 inFIG. 14A). Two clones are less phospho-specific (8B2D1) or non-specific(8B2D4) (FIG. 14A). Of the non-phospho-specific monoclonal antibodies,6B2E9 and 6B2G12, in particular, retained their non-specificity afterfurther subcloning (FIG. 14B). Data presented was obtained at 1:810dilution of culture supernatant.

FIGS. 15A-15B are western blots showing reactivity of the fourTau-386-408[P-Ser_(396, 404)] phospho-specific (FIG. 15A) andnon-phospho-specific (FIG. 15B) monoclonal antibody clones with brainhomogenates from the JNPL3 P301L mouse and wildtype (Wt) mouse. Of thefour phospho-specific clones, 4E6G7 shows the strongest reactivity,which is consistent with the ELISA results of FIG. 14A. In contrast withthe PHF-1 antibody that also recognizes the tan P-Ser_(396, 404)epitope, all clones react better with the JNPL3 P301L brain homogenatethan the Wt homogenate. The non-phospho-specific clones reacted faster,as expected, as most of tau is non-phosphorylated.

FIGS. 16A-16B illustrate the generation of monoclonal antibodies againstthe immunogenic tau peptide comprising amino acids 260-271 (SEQ ID NO:12) and containing phosphorylated serine 262 epitope. As shown in FIG.16A, a strong titer was generated against the immunogenTau260-271[P-Ser₂₆₂] (purple), but plasma antibodies recognized thenon-phospho peptide Tau260-271 as well (No-P; white). Eight stablephospho-specific clones were selected for further analysis (FIG. 16B).

FIG. 17 is a western blot showing the reactivity of the threephospho-specific Tau260-271[P-Ser₂₆₂] monoclonal antibody clones. The2C11 antibody clone recognizes a higher molecular weight band than theother phospho-specific clones and it does not distinguish betweenwildtype and P301L tissue. 5F7D10 and 5F7E9 are representatives of theother clones. Tau-5 recognizes total tau and binds to an epitope aroundamino acids 216-227 of tau. CP27 recognizes human but not mouse tau.

FIGS. 18A-18E are immunohistochemical photomicrographs showing thedetection of tau pathology using the 5F7D10 antibody clone in P301Ltangle mouse brain sections. The 5F7D10 monoclonal antibody shows stronghistological staining in the P301L brain section (FIG. 18A) compared tothe wildtype (FIG. 18B). The PHF1 antibody picked up tau pathology inthe same tangle mouse (FIG. 18C) although the pattern was different thanwith the 5F7D10 antibody, which is not surprising as they recognizedifferent tau epitopes. FIG. 18D is a magnified image of the boxedregion in FIG. 18A depicting neurons with aggregated tau. FIG. 18E is ahigher magnified image of tangle-like pathology detected with 5F7D10 ina different JNPL3 P301L mouse.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to a method ofpreventing or treating Alzheimer's disease or other tauopathy in asubject. This method involves administering, to the subject, any one ormore immunogenic tau peptides having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 2-75, or one or more antibodiesrecognizing an immunogenic tau epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 2-75 and 101-103 underconditions effective to treat or prevent Alzheimer's disease or othertauopathy in the subject.

As used herein a “tauopathy” encompasses any neurodegenerative diseasethat involves the pathological aggregation of the microtubule proteintau within the brain. Accordingly, in addition to both familial andsporadic Alzheimer's disease, other tauopathies that can be treatedusing the methods of the present invention include, without limitation,frontotemporal dementia, parkinsonism linked to chromosome 17 (FTDP-17),progressive supranuclear palsy, corticobasal degeneration, Pick'sdisease, progressive subcortical gliosis, tangle only dementia, diffuseneurofibrillary tangles with calcification, argyrophilic grain dementia,amyotrophic lateral sclerosis parkinsonism-dementia complex, dementiapugilistica, Down syndrome, Gerstmann-Straussler-Scheinker disease,Hallerworden-Spatz disease, inclusion body myositis, Creutzfeld-Jakobdisease, multiple system atropy, Niemann-Pick disease type C, prionprotein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, postencephalitic parkinsonism, and chronictraumatic encephalopathy.

Another aspect of the present invention is directed to a method ofpromoting clearance of tau aggregates from the brain of a subject. Thismethod involves administering, to the subject, any one or moreimmunogenic tau peptides having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-75, or one or more antibodiesrecognizing an immunogenic tau epitope comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-75 and 101-103 underconditions effective to promote clearance of tau aggregates from thebrain of the subject.

The clearance of tau aggregates includes clearance of neurofibrillarytangles and/or the pathological tau precursors to neurofibrillarytangles. Neurofibrillary tangles are often associated withneurodegenerative diseases including, for example, sporadic and familialAlzheimer's disease, amyotrophic lateral sclerosis, argyrophilic graindementia, dementia pugilistica, chronic traumatic encephalopathy,diffuse neurofibrillary tangles with calcification, Down syndrome,Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease,hereditary frontotemporal dementia, parkinsonism linked to chromosome 17(FTDP-17), inclusion body myositis, Creutsfeld-Jakob disease, multiplesystem atrophy, Niemann-Pick disease type C, Pick's disease, prionprotein cerebral amyloid angiopathy, sporadic corticobasal degeneration,progressive supranuclear palsy, subacute sclerosing panencephalitis,myotonic dystrophy, motor neuron disease with neurofibrillary tangles,tangle only dementia, and progressive subcortical gliosis.

Another aspect of the present invention is directed to a method ofslowing the progression of a tau-pathology related behavioral phenotypein a subject. This method involves administering, to the subject, anyone or more immunogenic tau peptides comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2-75, or one or moreantibodies recognizing an immunogenic tau epitope comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2-75 and101-103, under conditions effective to slow the tau-pathology relatedbehavioral phenotype in the subject.

As used herein, a tau-pathology related behavioral phenotype includes,without limitation, cognitive impairments, early personality change anddisinhibition, apathy, abulia, mutism, apraxia, perseveration,stereotyped movements/behaviors, hyperorality, disorganization,inability to plan or organize sequential tasks, selfishness/callousness,antisocial traits, a lack of empathy, halting agrammatic speech withfrequent paraphasic errors but relatively preserved comprehension,impaired comprehension and word-finding deficits, slowly progressivegait instability, retropulsions, freezing, frequent falls, non-levodoparesponsive axial rigidity, supranuclear gaze palsy, square wave jerks,slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia,cortical sensory loss, and tremor.

In accordance with the methods of the present invention, in oneembodiment, an immunogenic tau peptide or a combination of immunogenictau peptides are administered to a subject in need. Suitable immunogenictau peptide fragments of the tau protein contain one or more antigenicepitopes that mimic the pathological form of the tau protein. Exemplaryimmunogenic tau epitopes are phosphorylated at one or more amino acidsthat are phosphorylated in the pathological form of tau, but notphosphorylated in the normal or non-pathological form of tau.

In a preferred embodiment of the present invention, administration of animmunogenic tau peptide induces an active immune response in the subjectto the immunogenic tau peptide and to the pathological form of tau,thereby facilitating the clearance of related tau aggregates, slowingthe progression of tau-pathology related behavior and treating theunderlying tauopathy. In accordance with this aspect of the presentinvention, an immune response involves the development of a beneficialhumoral (antibody mediated) and/or a cellular (mediated byantigen-specific T cells or their secretion products) response directedagainst the immunogenic tau peptide.

The presence of a humoral immunological response can be determined andmonitored by testing a biological sample (e.g., blood, plasma, serum,urine, saliva feces, CSF or lymph fluid) from the subject for thepresence of antibodies directed to the immunogenic tau peptide. Methodsfor detecting antibodies in a biological sample are well known in theart, e.g., ELISA, Dot blots, SDS-PAGE gels or ELISPOT. The presence of acell-mediated immunological response can be determined by proliferationassays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte) assays which arereadily known in the art.

Isolated immunogenic tau peptides of the present invention include anyone of the amino acid sequences of SEQ ID NOs: 2-30 shown in Table 1below. Amino acid residues of each sequence which are phosphorylated areshown in bold and marked with asterisks. The names of the peptides inTable 1 correspond to the amino acid position of these peptides withinthe longest isoform of the human tau protein having the amino acidsequence of SEQ ID NO:1 as shown below.

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1               5                   10                  15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His            20                  25                  30Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu        35                  40                  45Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser    50                  55                  60Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val65                  70                  75                  80Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu                85                  90                  95Ile Pro Gln Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro            100                 105                 110Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val        115                 120                 125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly    130                 135                 140Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro145                 150                 155                 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro                165                 170                 175Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly            180                 185                 190Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser        195                 200                 205Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys    210                 215                 220Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225                 230                 235                 240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val                245                 250                 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly            260                 265                 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln        275                 280                 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly    290                 295                 300Ser Cal Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser305                 310                 315                 320Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln                325                 330                 335Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser            340                 345                 350Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn        355                 360                 365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala    370                 375                 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser385                 390                 395                 400Gly Aso Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser                405                 410                 415Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val            420                 425                 430Ser Ala Ser Leu Ala Lys Gln Gly Leu         435                 440

TABLE 1 Immunogenic Tau Peptides SEQ ID  NO: NAME SEQUENCE SEQ ID Tau210-216  SRT*PS*LP NO: 2 [P-Thr₂₁₂-Ser₂₁₄] SEQ ID  Tau260-264  IGS*TENO: 3 [P-Ser₂₆₂] SEQ ID  Tau229-237 VRT*PPKS*PS NO: 4 [P-Thr₂₃₁-Ser₂₃₅]SEQ ID  Tau394-406 YKS*PVVSGDTS*PR NO: 5 [P-Ser_(396,404)] SEQ ID Tau192-221 GDRSGYSSPGSPGTPGSRSRT* NO: 6 [P-Thr₂₁₂ Ser₂₁₄] PS*LPTPPTRSEQ ID  Tau192-221 GDRSGYSS*PGS*PGT* NO: 7 [P-Ser_(199,202,214,) PGSRSRT*PS*LPTPPTR Thr_(205,212)] SEQ ID  Tau192-221GDRSGYSS*PGSPGTPGSRSRT* NO: 8 [P-Ser_(199,214) PS*LPT*PPTRThr_(212,217)] SEQ ID  Tau192-221 GDRSGYSSPGS*PGT* NO: 9[P-Ser₂₀₂Thr₂₀₅] PGSRSRTPSLPTPPTR SEQ ID  Tau200-229 PGSPGTPGSRSRT*PS*NO: 10 [P-Thr₂₁₂-Ser₂₁₄] LPTPPTREPKKVAVV SEQ ID  Taw322-358CGS*LGNIHHKPGGGQV NO: 11 [P-Ser_(324,356)] EVKSEKLDFKDRVQSKIGS* LDSEQ ID  Tau260-271 IGS*TENLKHQPG NO: 12 [P-Ser₂₆₂] SEQ ID  Tau386-408TDHGAEIVYKS*PVVSGDTS* NO: 13 [P-Ser_(396,) Ser₄₀₄] PRHL SEQ ID  Tau48-71LQT*PTEDGSEEPG NO: 14 [P-Thr_(50,69)] SETSDAKST*PT SEQ ID  Tau111-115TPS*LE NO: 15 [P-Ser₁₁₃] SEQ ID  Tau151-155 IAT*PR NO: 16 [P-Thr₁₅₃]SEQ ID  Tau173-177 AKT*PP NO: 17 [P-Thr₁₇₅] SEQ ID  Tau203-219PGT*PGS*RS*RT* NO: 18 [P-Thr_(205,212,217)- PS*LPT*PP Ser_(208,210,214)SEQ ID  Tau233-237 PKS*PS NO: 19 [P-Thr₂₃₅] SEQ ID  Tau256-264VKS*KIGS*TE NO: 20 [P-Ser_(258,262)] SEQ ID  Tau287-291 VQS*KC NO: 21[P-Ser₂₈₉] SEQ ID  Tau354-358 IGS*LD NO: 22 [P-Ser₃₅₆] SEQ ID Tau398-416 VVS*GDT*SPRHLS*NVS* NO: 23 [P-S_(400,409,412,435)- S*T*GSThr_(403,414)] SEQ ID  Tau420-437 VDS*PQLAT*LADEVS* NO: 24[P-Ser_(422,433,435)- AS*LA Thr₄₂₇] SEQ ID  Tau200-204 PGS*P NO: 25[P-Ser₂₀₂] SEQ ID  Tau203-207 PGT*PG NO: 26 [P-Thr₂₀₅] SEQ ID Tau197-207 YSS*PGS*PGT*PG NO: 27 [P-Ser_(199,202)- Thr₂₀₅] SEQ ID Tau206-216 PGSRSRT*PS*LP NO: 28 [P-Thr₂₁₂- Ser₂₁₄] SEQ ID  Tau229-239VRT*PPKS*PSSA NO: 29 [P-Thr₂₃₁- Ser₂₃₅] SEQ ID  Tau179-188 PKT*PPS*S*GEPNO: 30 [P-Thr₁₈₁- Ser_(184,185)]

Variants and analogs of the above immunogenic peptides that induceand/or crossreact with antibodies to the preferred epitopes of tauprotein can also be used. Analogs, including allelic, species, andinduced variants, typically differ from naturally occurring peptides atone, two, or a few positions, often by virtue of conservativesubstitutions. Analogs typically exhibit at least 80 or 90% sequenceidentity with natural peptides. Some analogs also include unnaturalamino acids or modifications of N- or C-terminal amino acids at one,two, or a few positions.

In one embodiment of the present invention, variant immunogenic taupeptides are pseudo-phosphorylated peptides. The pseudo-phosphorylatedpeptides are generated by substituting one or more of the phosphorylatedserine, threonine, and tyrosine residues of the tau peptides with acidicamino acid residues such as glutamic acid and aspartic acid (Huang etal., “Constitutive Activation of Mekl by Mutation of SerinePhosphorylation Sites,” Proc. Natl. Acad. Sci. USA 91(19):8960-3 (1994),which is hereby incorporated by reference in its entirety). Exemplaryisolated immunogenic pseudo-phosphorylated tau peptides of the presentinvention are shown in Table 2 below. The position of the amino acidresidue substitutions is indicated in each sequence of Table 2 with an“X”, where X is an glutamic acid or aspartic acid residue substitution.

TABLE 2 Immunogenic Pseudo-Phosphorylated Tau Peptides SEQ ID NO: NAMESEQUENCE SEQ ID NO: 31 Tau210-216  SRXPXLP [T212X, S214X] SEQ ID NO: 32Tau260-264 IGXTE [S262X] SEQ ID NO: 33 Tau229-237 VRXPPKXPS[T231X, S235X] SEQ ID NO: 34 Tau392-406 YKXPVVSGDTXPR [S396X, S404X]SEQ ID NO: 35 Tau192-221 GDRSGYSSPGSPGTPGSRSRXPXLPTPPTR [T212X, S214X]SEQ ID NO: 36 Tau192-221 GDRSGYSXPGXPGXPGSRSRXPXLPTPPTR [S199X, S202X,S214X, T205X,  T212X] SEQ ID NO: 37 Tau192-221 GDRSGYSXPGSPGTPGSRSRXPXLPXPPTR [S199X, S214X, T212X, T217X]SEQ ID NO: 38 Tau192-221 GDRSGYSSPGXPGXPGSRSRTPSLPTPPTR [S202X. T205X]SEQ ID NO: 39 Tau200-229 PGSPGTPGSRSRXPXLPTPPTREPKKVAVV [T212X, S214X]SEQ ID NO: 40 Tau322-358 CGXLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGXLD[S324X, S356X] SEQ ID NO: 41 Tau260-271 IGXTENLKHQPG [S262X]SEQ ID NO: 42 Tau386-408 TDHGAEIVYKXPVVSGDTXPRHL [S396X, S404X]SEQ ID NO: 43 Tau48-71 LQXPTEDGSEEPGSETSDAKSXPT [T50X, T69X]SEQ ID NO: 44 Tau1111-115 TPXLE [S113X] SEQ ID NO: 45 Tau151-155 IAXPR[T153X] SEQ ID NO: 46 Tau173-177 AKXPP [T175X] SEQ ID NO: 47 Tau203-219PGXPGXRXRXPXLPXPP [T205X, T212X, T217X, S208X, S210X, S214X]SEQ ID NO: 48 Tau233-237 PKXPS [T235X] SEQ ID NO: 49 Tau256-264VKXKIGXTE [S258X, S262X] SEQ ID NO: 50 Tau287-291 VQXKC [S289X]SEQ ID NO: 51 Tau354-358 IGXLD [S356X] SEQ ID NO: 52 Tau398-416VVXGDXSPRHLXNVXXXGS [S400X, S409X, S412X, S413X, T403X, T414X]SEQ ID NO: 53 Tau420-437 VDXPQLAXLADEVXAXLA [S422X, S433X, S435X, T427X] SEQ ID NO: 54 Tau200-203 PGXP [S202X] SEQ ID NO: 55Tau203-207 PGXPG [T205X] SEQ ID NO: 56 Tau 133-162DGTGSDDKKAKGADGKXKIAXTPRGAAPPGQ [T149X, T153X] SEQ ID NO: 57 Tau 379-408RENAKAKTDHGAEIVYKXPVVSGDTXPRHL [S396X, S404X] SEQ ID NO: 58 Tau 192-221GDRSGYSXPGXPGXPGSRSRXPXLPTPPTR [S199X, S202X, S214X, T205X, T212X]SEQ ID NO: 59 Tau221-250 REPKKVAVVRXPPKXPSSAKSRLQTAPVPM [T231X, S235X]SEQ ID NO: 60 Tau184-213 XSGEPPKXGDRSQXXXPGXPGXPGXRXRX [S184X, S191X,Y197X, S198X, S199X, S202X, T205X, S208X, S210X, T212X] SEQ ID NO: 61Tau1-30 MAEPRQEFEVMEDHAGTXGLGDRKDQGGXT [Y18X, Y29X] SEQ ID NO: 62Tau30-60 TMHQDQEGDXDAGLKEXPLQXPXEDGXEEPG [T39X, S46X, T50X, T52X,  S56X]SEQ ID NO: 63 Tau60-90 GSETSDAKXXPXAEDVTAPLVDEGAPGKQAA [S68X, T69X T71X]SEQ ID NO: 64 Tau90-120 AAQPHXEIPEGXXAEEAGIGDTPXLEDEAAG [T95X, T101X,T102X, T113X] SEQ ID NO: 65 Tau120-150 GHVXQARMVSKXKDGTGSDDKKAKGADGKXK[T123x, S131X, T149X] SEQ ID NO: 66 Tau150-180KIATPRGAAPPGQKGQANATRIP AKXPPAPK [T175X] SEQ ID NO: 67 Tau180-210KXPPXXGEPPKSGDRSGXXXPGXPGXPGXRS [T181X, S184X, S185X, Y197X,S198X, S199X, S202X, T205X, S208X] SEQ ID NO: 68 Tau210-240SRXPXLPXPPTREPKKVAVVRXPPKXPXXAK [T212X, S214X, T217X, T231X,S235X, S237X, S238X] SEQ ID NO: 69 Tau240-270KSRLQTAPVPMPDLKNVKSKIGXTENLKHQP [S262X] SEQ ID NO: 70 Tau270-300PGGGKVQIINKKLDLSNVQSKCGXKDNIKHV [S293X] SEQ ID NO: 71 Tau300-330VPGGGSVQIVXKPVDLSKVTSKCGXLGNIHH [Y310, S324X] SEQ ID NO: 72 Tau330-360HKPGGGQVEVKSEKLDFKDRVQSKIGXLDNI [S356X] SEQ ID NO: 73 Tau360-390IXHVPGGGNKKIEXHKLTFRENAKAKXDHGA [T361X, T373X, T386X] SEQ ID NO: 74Tau390-420 AEIVXKXPVVXGDXXPRHLXNVXXTGSIDMV [Y394X, S396X, S400X, T403X,S404X, S409X, S412X, S413X] SEQ ID NO: 75 Tau411-441VXXTGSIDMVDXPQLATLADEVSASLAKQGL [S412X, S413X, S422X]

Each tau peptide of the present invention, i.e., SEQ ID NOs: 2-75 and87-88 (Table 3 below) is preferably acetylated on the N-terminus andamidated on the C-terminus to more closely resemble the same internalamino acids of the full length tau protein. The tau peptides of thepresent invention can also contain one or more D-amino acid residues toenhance the stability of the peptide. These D-amino acids can be in thesame order as the L-form of the peptide or assembled in a reverse orderfrom the L-form sequence to maintain the overall topology of the nativesequence (Ben-Yedidia et al., “A Retro-Inverso Peptide Analogue ofInfluenza Virus Hemagglutinin B-cell Epitope 91-108 Induces a StrongMucosal and Systemic Immune Response and Confers Protection in Miceafter Intranasal Immunization,” Mol Immunol. 39:323 (2002); Guichard, etal., “Antigenic Mimicry of Natural L-peptides withRetro-Inverso-Peptidomimetics,” PNAS 91:9765-9769 (1994); Benkirane, etal., “Antigenicity and Immunogenicity of Modified Synthetic PeptidesContaining D-Amino Acid Residues,” J. Bio. Chem. 268(35):26279-26285(1993), which are hereby incorporated by reference in their entirety).

Each of the above peptide sequences may be linked to an immunogeniccarrier molecule to enhance its immunogenicity. Suitable immunogeniccarrier molecules include, but are not limited to, helper T-cellepitopes, such as tetanus toxoid (e.g., the P2 and P30 epitopes),Hepatitis B surface antigen, cholera toxin B, toxoid, diphtheria toxoid,measles virus F protein, Chlamydia trachomatis major outer membraneprotein, Plasmodium falciparum circumsporozite T. P. falciparum CSantigen, Schistosoma mansoni triose phosphate isomerase, Bordetellapertussis, Clostridium tetani, Pertusaria trachythallina, Escherichiacoli TraT, and Influenza virus hemagluttinin (HA) (see U.S. Pat. No.6,906,169 to Wang; U.S. Patent Application Publication No. 20030068325to Wang, and WO/2002/096350 to Wang, which are hereby incorporated byreference in their entirety). In a preferred embodiment of the presentinvention, the T-helper cell epitope is the tetanus toxin 947-967 (P30)epitope having an amino acid sequence of FNNFTVSFWLRVPKVSASHLE (SEQ IDNO: 76). In another embodiment, the T-helper cell epitope is the tetanustoxin 830-843 (P2) epitope having an amino acid sequence ofQYIKANSKFIGIT (SEQ ID NO: 77).

The immunogenic tau peptides of the present invention can be linked tothe immunogenic carrier molecule using a short amino acid linkersequence. In a preferred embodiment of the present invention, a GPSL(SEQ ID NO: 78) linker sequence is used to link the immunogenic taupeptide to the immunogenic carrier molecule. Other suitable linkersequences include glycine-rich (e.g. G₃₋₅) or serine-rich (e.g., GSG,GSGS (SEQ ID NO: 79), GSGSG (SEQ ID NO: 80), GS_(N)G) linker sequencesor flexible immunoglobulin linkers as disclosed in U.S. Pat. No.5,516,637 to Huang et al, which is hereby incorporated by reference inits entirety.

Alternatively, the immunogenic tau peptides of the present invention canbe linked to the immunogenic carrier molecule using chemicalcrosslinking. Techniques for linking a peptide immunogen to animmunogenic carrier molecule include the formation of disulfide linkagesusing N-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP) andsuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (ifthe peptide lacks a sulfhydryl group, this can be provided by additionof a cysteine residue). These reagents create a disulfide linkagebetween themselves and peptide cysteine residues on one protein, and anamide linkage through the epsilon-amino on a lysine, or other free aminogroup in other amino acids. A variety of such disulfide/amide-formingagents are described by Jansen et al., “Immunotoxins: Hybrid MoleculesCombining High Specificity and Potent Cytotoxicity,” Immun Res62:185-216 (1982), which is incorporated by reference in its entirety.Other bifunctional coupling agents form a thioether rather than adisulfide linkage. Many of these thio-ether-forming agents arecommercially available and include reactive esters of 6-maleimidocaproicacid, 2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Immunogenic tau peptides of the present invention can be synthesized bysolid phase peptide synthesis or recombinant expression systems.Automatic peptide synthesizers are commercially available from numeroussuppliers, such as Applied Biosystems (Foster City, Calif.). Recombinantexpression systems can include bacteria, such as E. coli, yeast, insectcells, or mammalian cells. Procedures for recombinant expression aredescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual(C.S.H.P. Press, NY 2d ed., 1989), which is hereby incorporated byreference in its entirety.

The immunogenic tau peptides of the present invention can beadministered alone or in combination with other immunogenic tau peptidesof the present invention to a subject in need. In one embodiment, animmunogenic tau peptide of the present invention is administered incombination with one or more immunogenic tau peptides shown in Table 3below as disclosed in U.S. Patent Application Publication No.20080050383 to Sigurdsson, which is hereby incorporated by reference inits entirety. The names of the peptides in Table 3 correspond to theamino acid position of these peptides within the longest isoform of thetau protein having the amino acid sequence of SEQ ID NO:1. Amino acidresidues of each sequence which are phosphorylated are shown in bold andmarked with asterisks.

TABLE 3 Immunogenic Tau Peptide  Sequences for Combined AdministrationSEQ ID  NO: NAME SEQUENCE SEQ ID  Tau 133-162DGTGSDDKKAKGADGKTKIATPRGAAPPGQ NO: 81 SEQ ID  Tau 379-408RENAKAKTDHGAEIVYKS*PVVSGDTS*PRHL NO: 82 [P-Ser_(396,404)] SEQ ID Tau 192-221 GDRSGYSS*PGS*PGT*PGSRSRT*PA*LPTPPTR NO: 83[P-Ser_(199,202,214),- Thr_(205,212)] SEQ ID  Tau221-250REPKKVAVVRT*PPKS*PSSAKSRLQTAPVPM NO: 84 [P-Thr₂₃₁,-Ser₂₃₅] SEQ ID Tau184-213 SSGEPPKSGDRSQYSSPGSPGTPGSRSRT NO: 85 SEQ ID  Tau1-30MAEPRQEFEVMEDHAGTY*GLGDRKDQGGY*T NO: 86 [P-Tyr_(18,29)] SEQ ID  Tau30-60TMHQDQEGDTDAGLKESPLQTPTEDGSEEPG NO: 87 SEQ ID  Tau60-90GSETSDAKSTPTAEDVTAPLVDEGAPGKQAA NO: 88 SEQ ID  Tau90-120AAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG NO: 89 SEQ ID  Tau120-150GHVTQARMVSKSKDGTGSDDKKAKGADGKTK NO: 90 SEQ ID  Tau150-180KIATPRGAAPPGQKGQANATRIPAKT*PPAPK NO: 91 [P-Thr₁₇₅] SEQ ID  Tau180-210KT*PPS*S*GEPPKSGDRSGY*S*S*PGS*PGT*PGS*RS NO: 92 [P-Thr_(181,205),-Ser_(184,185,198,199,202,208),- Tyr₁₉₇] SEQ ID  Tau210-240SRT*PS*LPT*PPTREPKKVAVVRT*PPKS*PS*S*AK NO: 93 [P-Thr_(212,217,231),-Ser_(214,235,237,238)] SEQ ID  Tau240-270KSRLQTAPVPMPDLKNVKSKIGS*TENLKHQP NO: 94 [P-Ser₂₆₂] SEQ ID  Tau270-300PGGGKVQIINKKLDLSNVQSKCGS*KDNIKHV NO: 95 [P-Ser₂₉₃] SEQ ID  Tau300-330VPGGGSVQIVY*KPVDLSKVTSKCGS*LGNIHH NO: 96 [P-Tyr₃₁₀, Ser₃₂₄] SEQ ID Tau330-360 HKPGGGQVEVKSEKLDFKDRVQSKIGS*LDNI NO: 97 [P-Ser₃₅₆] SEQ ID Tau360-390 ITHVPGGGNKKIETHKLTPRENAKAKTDHGA NO: 98 SEQ ID  Tau390-420ARIVY*KS*PVVS*GDT*S*PRHLS*NVS*S*TGSIDMV NO: 99 [P-Tyr₃₉₄,Ser_(396,400,404,409,412,413), Thr₄₀₃] SEQ ID  Tau411-441VS*S*TGSIDMVDS*PQLATLADEVSASLAKQGL NO: 100 [P-Ser_(412,413,422)]

The immunogenic tau peptides of the present invention can beadministered in combination with a suitable adjuvant to achieve thedesired immune response in the subject. Suitable adjuvants can beadministered before, after, or concurrent with administration of theimmunogenic tau peptide of the present invention. Preferred adjuvantsaugment the intrinsic response to an immunogen without causingconformational changes in the immunogen that affect the qualitative formof the response.

A preferred class of adjuvants is the aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, and aluminum sulfate. Suchadjuvants can be used with or without other specific immunostimulatingagents, such as 3 De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP,polymeric or monomeric amino acids, such as polyglutamic acid orpolylysine. Such adjuvants can be used with or without other specificimmunostimulating agents, such as muramyl peptides (e.g.N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wallcomponents. Oil-in-water emulsions include MF59 (see WO 90/14837 to VanNest et al., which is hereby incorporated by reference in its entirety),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer; SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion; and the Ribi™ adjuvant system (RAS) (RibiImmunoChem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80, andone or more bacterial cell wall components selected from the groupconsisting of monophosphoryllipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Otheradjuvants include Complete Freund's Adjuvant (CFA), Incomplete Freund'sAdjuvant (IFA), and cytokines, such as interleukins (IL-1, IL-2, andIL-12), macrophage colony stimulating factor (M-CSF), and tumor necrosisfactor (TNF).

The choice of an adjuvant depends on the stability of the immunogenicformulation containing the adjuvant, the route of administration, thedosing schedule, the efficacy of the adjuvant for the species beingvaccinated, and, in humans, a pharmaceutically acceptable adjuvant isone that has been approved or is approvable for human administration bypertinent regulatory bodies. For example, alum, MPL or IncompleteFreund's adjuvant (Chang et al., Advanced Drug Delivery Reviews32:173-186 (1998), which is hereby incorporated by reference in itsentirely) alone or optionally all combinations thereof are suitable forhuman administration.

Another aspect of the present invention relates to a pharmaceuticalcomposition containing one or more of the immunogenic tau peptidesdescribed supra and a pharmaceutical carrier (describe infra). Thepharmaceutical composition may contain a mixture of the same immunogenictau peptide. Alternatively, the pharmaceutical composition contains amixture of one or more different immunogenic tau peptides of the presentinvention. In a preferred embodiment, pharmaceutical compositions of thepresent invention contain one or more suitable adjuvants as describedsupra.

In another embodiment of the present invention, an antibody recognizingone or more of the immunogenic tau epitopes of the present invention isadministered to a subject in need. Suitable antibodies of the presentinvention encompass any immunoglobulin molecule that specifically bindsto an immunogenic tau epitope comprising any one of amino acid sequencesof SEQ ID NOs: 2-75 and 101-103. In a preferred embodiment, an antibodyof the present invention recognizes and binds to an epitope specific forthe pathological form of tau and has little to no crossreactivity withthe normal tau protein or a non-tau protein.

As described herein, monoclonal antibodies recognizing the immunogenictau epitopes comprising SEQ ID NO: 13 (Tau 386-408 [P-Ser_(396,404)])and SEQ ID NO: 12 (Tau 260-271[P-Ser₂₆₂]) have been generated. Theseantibodies are phospho-specific and, therefore, specific for thepathological tau forms having little to no crossreactivity to the normaltau protein.

In addition to the antibodies recognizing phosphorylated pathologicalepitopes of the tau protein, the present invention is also directed toantibodies that preferentially recognize pathological tau fragmentsinvolved in promoting neuronal toxicity and/or seeding tau aggregation.For example, caspase cleavage of tau, preferentially at aspartateresidue 421 (D421) of the tau protein, creates a truncated molecule thatcolocalizes with tangles and correlates with the progression inAlzheimer's disease and in animal models of tauopathy (see Calignon etal., “Caspase Activation Precedes and Leads to Tangles,” Nature464:120-1205 (2010), which is hereby incorporated by reference in itsentirety). An antibody directed to the free D421 end of the cleaved tauprotein would be specific for, and facilitate the removal of,pathological tau but not normal tau. Accordingly, the present inventionis directed to an antibody, preferably a monoclonal antibody, directedto D421 on the free C-terminus of a cleaved pathological tau protein,that is not present in the normal tau protein. In one embodiment of thepresent invention, the antibody is generated using the methods describedherein with an immunogenic tau peptide comprising an amino acid sequenceof HLSNVSSTGSIDMVD (SEQ ID NO: 101).

Truncation of tau at glutamic acid residue 391 (E391) is also associatedwith neurofibrillary tangle formation in the brains of Alzheimer'sdisease patients (Basurto-islas et al. “Accumulation of AsparticAcid⁴²¹—and Glutamic Acid³⁹¹—Cleaved Tau in Neurofibrillary TanglesCorrelates with Progression in Alzheimer Disease,” J Neuropathol ExpNeurol 67:470-483 (2008), which is hereby incorporated by reference inits entirety). Accordingly, the present invention is also directed to anantibody, preferably a monoclonal antibody, directed to E391 on the freeC-terminus of a cleaved pathological tau protein, that is not present inthe normal tau protein. In one embodiment of the present invention, theantibody is generated using the methods described herein with animmunogenic tau peptide comprising an amino acid sequence ofRENAKAKTDHGAE (SEQ ID NO: 102)

Calpain-1 also mediates the cleavage of tau, generating a toxic 17 kDatau fragment that promotes Aβ-induced neurotoxicity (Park et al., “TheGeneration of a 17 kDa Neurotoxic Fragment: An Alternative Mechanism bywhich Tau Mediates β-Amyloid-Induced Neurodegeneration,” J. Neurosci25(22):5365-75 (2005), which is hereby incorporated by reference in itsentirety). Accordingly, an embodiment of the present invention is alsodirected to an antibody, preferably a monoclonal antibody, specificallyrecognizing the free N- and/or free C-terminus of this toxic taufragment, but not the normal tau protein, comprising amino acid residues45-230 of tau (SEQ ID NO:1) shown as SEQ ID NO:103 below.

Glu Ser Pro Leu Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly1               5                   10                  15Ser Glu Thr Ser Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr            20                  25                  30Ala Pro Leu Val Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln        35                  40                  45Pro His Thr Glu Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile    50                  55                  60Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln65                  70                  75                  80Ala Arg Met Val Ser Lys Ser Lys Asp Gly The Gly Ser Asp Asp Lys                85                  90                  95Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly            100                 105                 110Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro        115                 120                 125Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro    130                 135                 140Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly145                 150                 155                 160Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr                165                 170                 175Arg Glu Pro Lys Lys Val Ala Val Val Arg            180                 185

As used herein, the term “antibody” includes intact immunoglobulinsderived from natural sources or from recombinant sources, as well asimmunoreactive portions (i.e., antigen binding portions) of intactimmunoglobulins. The antibodies of the present invention may exist in avariety of forms including, for example, polyclonal antibodies,monoclonal antibodies, intracellular antibodies (“intrabodies”),antibody fragments (e.g., Fv, Fab and F(ab)2), as well as single chainantibodies (scFv), chimeric antibodies and humanized antibodies (EdHarlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL (ColdSpring Harbor Laboratory Press, 1999): Houston et al., “ProteinEngineering of Antibody Binding Sites: Recovery of Specific Activity inan Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,”Proc Natl Acad Sci USA 85:5879-5883 (1988); Bird et al, “Single-ChainAntigen-Binding Proteins,” Science 242:423-426 (1988)).

Methods for monoclonal antibody production may be carried out using thetechniques described herein or others well-known in the art (MONOCLONALANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS (Mary A.Ritter and Heather M. Ladyman eds., 1995), which is hereby incorporatedby reference in its entirety). Generally, the process involves obtainingimmune cells (lymphocytes) from the spleen of a mammal which has beenpreviously immunized with the antigen of interest (i.e., an immunogenictau peptide) either in vivo or in vitro. Exemplary tau peptides aredescribed supra. For generating monoclonal antibodies using the taupeptides of SEQ ID NOs: 2-75 or tau peptides of SEQ ID NOs: 101-103, acysteine residue may be added to the N- or C-terminus of each sequenceto facilitate linkage of a carrier protein that will enhance antibodyproduction upon immunization. Suitable carrier proteins include, withoutlimitation keyhole limpet hemocyanine, blue carrier immunogenic protein(derived from Concholepas concholepas), bovine serum albumin (BSA),ovalbumin, and cationized BSA.

The antibody-secreting lymphocytes are fused with myeloma cells ortransformed cells, which are capable of replicating indefinitely in cellculture, thereby producing an immortal, immunoglobulin-secreting cellline. Fusion with mammalian myeloma cells or other fusion partnerscapable of replicating indefinitely in cell culture is achieved bystandard and well-known techniques, for example, by using polyethyleneglycol (PEG) or other fusing agents (Milstein and Kohler, “Derivation ofSpecific Antibody-Producing Tissue Culture and Tumor Lines by CellFusion,” Eur J Immunol 6:511 (1976), which is hereby incorporated byreference in its entirety). The immortal cell line, which is preferablymurine, but may also be derived from cells of other mammalian species,is selected to be deficient in enzymes necessary for the utilization ofcertain nutrients, to be capable of rapid growth, and have good fusioncapability. The resulting fused cells, or hybridomas, are cultured, andthe resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody.

Alternatively, monoclonal antibodies can be made using recombinant DNAmethods as described in U.S. Pat. No. 4,816,567 to Cabilly et al, whichis hereby incorporated by reference in its entirety. The polynucleotidesencoding a monoclonal antibody are isolated from mature B-cells orhybridoma cells, for example, by RT-PCR using oligonucleotide primersthat specifically amplify the genes encoding the heavy and light chainsof the antibody. The isolated polynucleotides encoding the heavy andlight chains are then cloned into suitable expression vectors, whichwhen transfected into host cells such as E. coli cells, simian COScells, Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, monoclonal antibodies aregenerated by the host cells. Also, recombinant monoclonal antibodies orfragments thereof of the desired species can be isolated from phagedisplay libraries (McCafferty et al., “Phage Antibodies: FilamentousPhage Displaying Antibody Variable Domains,” Nature 348:552-554 (1990);Clackson et al., “Making Antibody Fragments using Phage DisplayLibraries,” Nature 352:624-628 (1991); and Marks et al., “By-PassingImmunization. Human Antibodies from V-Gene Libraries Displayed onPhage,” J. Mol. Biol. 222:581-597 (1991), which are hereby incorporatedby reference in their entirety).

The polynucleotide(s) encoding a monoclonal antibody can further bemodified using recombinant DNA technology to generate alternativeantibodies. For example, the constant domains of the light and heavychains of a mouse monoclonal antibody can be substituted for thoseregions of a human antibody to generate a chimeric antibody.Alternatively, the constant domains of the light and heavy chains of amouse monoclonal antibody can be substituted for a non-immunoglobulinpolypeptide to generate a fusion antibody. In other embodiments, theconstant regions are truncated or removed to generate the desiredantibody fragment of a monoclonal antibody. Furthermore, site-directedor high-density mutagenesis of the variable region can be used tooptimize specificity and affinity of a monoclonal antibody.

The monoclonal antibody of the present invention can be a humanizedantibody. Humanized antibodies are antibodies that contain minimalsequences from non-human (e.g. murine) antibodies within the variableregions. Such antibodies are used therapeutically to reduce antigenicityand human anti-mouse antibody responses when administered to a humansubject.

An antibody can be humanized by substituting the complementaritydetermining region (CDR) of a human antibody with that of a non-humanantibody (e.g. mouse, rat, rabbit, hamster, etc.) having the desiredspecificity, affinity, and capability (Jones et al., “Replacing theComplementarity-Determining Regions in a Human Antibody With Those Froma Mouse,” Nature 321:522-525 (1986); Riechmann et al., “Reshaping HumanAntibodies for Therapy,” Nature 332:323-327 (1988): Verhoeyen et al.,“Reshaping Human Antibodies: Grafting an Antilysozyme Activity,” Science239:1534-1536 (1988), which are hereby incorporated by reference intheir entirety). The humanized antibody can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the replaced non-human residues to refine and optimizeantibody specificity, affinity, and/or capability.

Human antibodies can be produced using various techniques known in theart. Immortalized human B lymphocytes immunized in vitro or isolatedfrom an immunized individual that produce an antibody directed against atarget antigen can be generated (See e.g. Reisfeld t al., MONOCLONALANTIBODIES AND CANCER THERAPy 77 (Alan R. Liss ed., 1985) and U.S. Pat.No. 5,750,373 to Garrard, which are hereby incorporated by reference intheir entirety). Alternatively, the human antibody can be selected froma phage library, where that phage library expresses human antibodies(Vaughan et al., “Human Antibodies with Sub-Nanomolar AffinitiesIsolated from a Large Non-immunized Phage Display Library,” NatureBiotechnology, 14:309-314 (1996); Sheets et al., “Efficient Constructionof a Large Nonimmune Phage Antibody Library: The Production ofHigh-Affinity Human Single-Chain Antibodies to Protein Antigens,” Proc.Natl. Acad. Sci. U.S.A. 95:6157-6162 (1998); Hoogenboom et al.,“By-passing Immunization. Human Antibodies From Synthetic Repertoires ofGermline VH Gene Segments Rearranged In Vitro,” J Mol Biol 227:381-8(1992); Marks et al. “By-passing Immunization. Human Antibodies fromV-gene Libraries Displayed on Phage,” J Mol Biol 222:581-97 (1991),which are hereby incorporated by reference in their entirety). Humanantibodies can also be made in transgenic mice containing humanimmunoglobulin loci that are capable upon immunization of producing thefull repertoire of human antibodies in the absence of endogenousimmunoglobulin production. This approach is described in U.S. Pat. No.5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg et al.;U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No. 5,625,126 toLonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et al.; and U.S. Pat.No. 5,661,016 to Lonberg et al., which are hereby incorporated byreference in their entirety

Procedures for raising polyclonal antibodies are also well known in theart. Typically, such antibodies can be raised by administering thepeptide containing the epitope of interest (i.e. any tau peptideselected from the group consisting of SEQ ID NOs: 2-75 or SEQ ID NOs:101-103) subcutaneously to New Zealand white rabbits which have beenbled to obtain pre-immune serum. The antigens can be injected incombination with an adjuvant. The rabbits are bled approximately everytwo weeks after the first injection and periodically boosted with thesame antigen three times every six weeks. Polyclonal antibodies arerecovered from the serum by affinity chromatography using thecorresponding antigen to capture the antibody. This and other proceduresfor raising polyclonal antibodies are disclosed in Ed Harlow and DavidLane. USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring HarborLaboratory Press, 1988), which is hereby incorporated by reference inits entirety.

In addition to whole antibodies, the present invention encompassesbinding portions of such antibodies. Such binding portions include themonovalent Fab fragments. Fv fragments (e.g., single-chain antibody,scFv), and single variable V_(H) and V_(L) domains, and the bivalentF(ab′)₂ fragments, Bis-scFv, diabodies, triabodies, minibodies, etc.These antibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL(Cold Spring Harbor Laboratory, 1988), which are hereby incorporated byreference in their entirety, or other methods known in the art.

Also suitable for use in the present invention are antibody fragmentsengineered to bind to intracellular proteins, i.e. intrabodies.Intrabodies directed to an immunogenic tau epitope comprising any one ofSEQ ID NOs: 2-75 of SEQ ID NOs: 101-103 can prevent pathological tauaggregation and accumulation within neurons or glial cells and/orfacilitate aggregate clearance. The application of intrabody technologyfor the treatment of neurological disorders, including tauopathies, isreviewed in Miller et al., “Intrabody Applications in NeurologicalDisorders: Progress and Future Prospects,” Mol Therapy 12:394-401(2005), which is hereby incorporated by reference in its entirety.

Intrabodies are generally obtained by selecting a single variable domainfrom variable regions of an antibody having two variable domains (i.e.,a heterodimer of a heavy chain variable domain and a light chainvariable domain). Single chain Fv fragments, Fab fragments, ScFv-Ckfusion proteins, single chain diabodies, V_(H)-C_(H)1 fragments, andeven whole IgG molecules are suitable formats for intrabody development(Kontermann R. E., “Intrabodies as Therapeutic Agents,” Methods34:163-70 (2004), which is here by incorporated by reference in itsentirety).

Intrabodies having antigen specificity for a pathological tau proteinepitope can be obtained from phage display, yeast surface display, orribosome surface display. Methods for producing libraries of intrabodiesand isolating intrabodies of interest are further described in U.S.Published Patent Application No. 20030104402 to Zauderer and U.S.Published Patent Application No. 20050276800 to Rabbitts, which arehereby incorporated by reference in their entirety. Methods forimproving the stability and affinity binding characteristics ofintrabodies are described in WO2008070363 to Zhenping andContreras-Martinez et al., “Intracellular Ribosome Display via SecMTranslation Arrest as a Selection for Antibodies with Enhanced CytosolicStability,” J Mol Biol 372(2):513-24 (2007), which are herebyincorporated by reference in their entirety.

It may further be desirable, especially in the case of antibodyfragments, to modify the antibody in order to increase its serumhalf-life. This can be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment by mutationof the appropriate region in the antibody fragment or by incorporatingthe epitope into a peptide tag that is then fused to the antibodyfragment at either end or in the middle (e.g., by DNA or peptidesynthesis).

Antibody mimics are also suitable for use in accordance with the presentinvention. A number of antibody mimics are known in the art including,without limitation, those known as monobodies, which are derived fromthe tenth human fibronectin type III domain (¹⁰Fn3) (Koide et al., “TheFibronectin Type III Domain as a Scaffold for Novel Binding Proteins,” JMol Biol 284:1141-1151 (1998); Koide et al., “Probing ProteinConformational Changes in Living Cells by Using Designer BindingProteins: Application to the Estrogen Receptor,” Proc Natl Acad Sci USA99:1253-1258 (2002), each of which is hereby incorporated by referencein its entirety), and those known as affibodies, which are derived fromthe stable alpha-helical bacterial receptor domain Z of staphylococcalprotein A (Nord et al., “Binding Proteins Selected from CombinatorialLibraries of an alpha-helical Bacterial Receptor Domain,” NatureBiotechnol 15(8):772-777 (1997), which is hereby incorporated byreference in its entirety).

The present invention is further directed to pharmaceutical compositionscontaining the one or more antibodies recognizing the immunogenic taupeptides of the present invention as described supra. Thispharmaceutical composition may contain a mixture of the same antibodiesrecognizing the same tau epitope. Alternatively, the pharmaceuticalcomposition may contain a mixture of one or more antibodies recognizingone or more different tau epitopes. The pharmaceutical composition ofthe present invention further contains a pharmaceutically acceptablecarrier or other pharmaceutically acceptable components as describedinfra.

The pharmaceutical compositions of the present invention containing theimmunogenic tau peptides or antibodies recognizing the immunogenic taupeptides, contain, in addition to the active therapeutic agent, avariety of other pharmaceutically acceptable components (see Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.,1980), which is hereby incorporated by reference in its entirety). Thepreferred formulation of the pharmaceutical composition depends on theintended mode of administration and therapeutic application. Thecompositions can include pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, non-immunogenic stabilizers, and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

The pharmaceutical compositions of the present invention can furtherinclude a suitable delivery vehicle. Suitable delivery vehicles include,but are not limited to viruses, bacteria, biodegradable microspheres,microparticles, nanoparticles, liposomes, collagen minipellets, andcochleates.

In one embodiment of the present invention, the delivery vehicle is avirus or bacteria and the immunogenic tau peptide is presented by avirus or bacteria as part of an immunogenic composition. In accordancewith this embodiment of the invention, a nucleic acid molecule encodingthe immunogenic peptide is incorporated into a genome or episome of thevirus or bacteria. Optionally, the nucleic acid molecule is incorporatedin such a manner that the immunogenic peptide is expressed as a secretedprotein or as a fusion protein with an outer surface protein of a virusor a transmembrane protein of bacteria so that the peptide is displayed.Viruses or bacteria used in such methods should be nonpathogenic orattenuated. Suitable viruses include adenovirus, HSV, Venezuelan equineencephalitis virus and other alpha viruses, vesicular stomatitis virus,and other rhabdo viruses, vaccinia and fowl pox. Suitable bacteriainclude Salmonella and Shigella. Fusion of an immunogenic peptide toHBsAg of HBV is particularly suitable.

In another embodiment of the present invention, the pharmaceuticalcomposition contains a liposome delivery vehicle. Liposomes are vesiclescomprised of one or more concentrically ordered lipid bilayers whichencapsulate an aqueous phase. An immunogenic tau peptide or antibodyraised against an immunogenic tau peptide of the present invention canbe surface bound, encapsulated, or associated with the membrane of theliposome vehicle. Various types of liposomes suitable for vaccinedelivery of the tau peptides are known in the art (see e.g., Hayashi etal., “A Novel Vaccine Delivery System Using Immunopotentiating FusogenicLiposomes,” Biochem Biophys Res Commun 261(3):824-28 (1999) and U.S.Patent Publication No. 20070082043 to Michaeli et al., which are herebyincorporated by reference in their entirety). Other methods forpreparing liposomes for use in the present invention include thosedisclosed in Bangham et al., “Diffusion of Univalent Ions Across theLamellae of Swollen Phospholipids,” J. Mol. Biol. 13:238-52 (1965); U.S.Pat. No. 5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S.Pat. No. 5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau &Kaneda; and U.S. Pat. No. 5,059,421 to Loughrey et al., which are herebyincorporated by reference in their entirety.

In another embodiment of the present invention, a nucleic acid moleculeencoding an immunogenic tau peptide or a tau antibody of the presentinvention is administered using a gene therapy delivery system. Suitablegene therapy vectors include, without limitation, adenoviral vectors,adeno-associated viral vectors, retroviral vectors, lentiviral vectors,and herpes viral vectors.

Adenoviral viral vector delivery vehicles can be readily prepared andutilized as described in Berkner, “Development of Adenovirus Vectors forthe Expression of Heterologous Genes,” Biotechniques 6:616-627 (1988)and Rosenfeld et al., “Adenovirus-Mediated Transfer of a RecombinantAlpha 1-Antitrypsin Gene to the Lung Epithelium In Vivo,” Science252:431-434 (1991), WO 93/07283 to Curiel et al., WO 93/06223 toPerricaudet et al., and WO 93/07282 to Curiel et al., which are herebyincorporated by reference in their entirety. Adeno-associated viraldelivery vehicles can be constructed and used to deliver a nucleic acidencoding a tau antibody of the present invention to cells as describedin Shi et al., “Therapeutic Expression of an Anti-Death Receptor-5Single-Chain Fixed Variable Region Prevents Tumor Growth in Mice,”Cancer Res. 66:11946-53 (2006); Fukuchi et al. “Anti-Aβ Single-ChainAntibody Delivery via Adeno-Associated Virus for Treatment ofAlzheimer's Disease,” Neurobiol. Dis. 23:502-511 (2006); Chatterjee etal., “Dual-Target Inhibition of HIV-1 In Vitro by Means of anAdeno-Associated Virus Antisense Vector,” Science 258:1485-1488 (1992);Ponnazhagan et al., “Suppression of Human Alpha-Globin Gene ExpressionMediated by the Recombinant Adeno-Associated Virus 2-Based AntisenseVectors,” J. Exp. Med. 179:733-738 (1994); and Zhou et al.,“Adeno-associated Virus 2-Mediated Transduction and ErythroidCell-Specific Expression of a Human Beta-Globin Gene,” Gene Ther.3:223-229 (1996), which are hereby incorporated by reference in theirentirety. In vivo use of these vehicles is described in Flotte et al.,“Stable in Vivo Expression of the Cystic Fibrosis TransmembraneConductance Regulator With an Adeno-Associated Virus Vector,” Proc.Nat'l. Acad. Sci. 90:10613-10617 (1993) and Kaplitt et al., “Long-TermGene Expression and Phenotypic Correction Using Adeno-Associated VirusVectors in the Mammalian Brain,” Nature Genet. 8:148-153 (1994), whichare hereby incorporated by reference in their entirety. Additional typesof adenovirus vectors are described in U.S. Pat. No. 6,057,155 toWickham et al.; U.S. Pat. No. 6,033,908 to Bout et al.; U.S. Pat. No.6,001,557 to Wilson et al.; U.S. Pat. No. 5,994,132 to Chamberlain etal.; U.S. Pat. No. 5,981,225 to Kochanek et al.; U.S. Pat. No. 5,885,808to Spooner et al.; and U.S. Pat. No. 5,871,727 to Curiel, which arehereby incorporated by reference in their entirety.

Retroviral vectors which have been modified to form infectivetransformation systems can also be used to deliver nucleic acidmolecules encoding a desired peptide or antibody to a target cell. Onesuch type of retroviral vector is disclosed in U.S. Pat. No. 5,849,586to Kriegler et al., which is hereby incorporated by reference.

Gene therapy vectors carrying a nucleic acid molecule encoding theimmunogenic tau peptide or tau antibody are administered to a subjectby, for example, intravenous injection, local administration (U.S. Pat.No. 5,328,470 to Nabel et al., which is hereby incorporated by referencein its entirety) or by stereotactic injection (see e.g., Chen et al.,“Gene Therapy for Brain Tumors: Regression of Experimental Gliomas byAdenovirus Mediated Gene Transfer in Vivo,” Proc. Nat'l. Acad. Sci. USA91:3054-3057 (1994), which is hereby incorporated by reference in itsentirety). The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded.

In carrying out the methods of the present invention, it is preferableto select a subject having or at risk of having Alzheimer's disease orother tauopathy, a subject having tau aggregates in the brain, or asubject exhibiting a tangle related behavioral phenotype prior toadministering the immunogenic peptides or antibodies of the presentinvention. Subjects amenable to treatment include individuals at risk ofdisease but not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease. Therefore, the presentmethods can be administered prophylactically to the general populationwithout the need for any assessment of the risk of the subject patient.The present methods are especially useful for individuals who do have aknown genetic risk of Alzheimer's disease. Such individuals includethose having relatives who have experienced this disease, and thosewhose risk is determined by analysis of genetic or biochemical markers.Genetic markers of risk toward Alzheimer's disease include mutations inthe APP gene, particularly mutations at position 717 and positions 670and 671 referred to as the Hardy and Swedish mutations, respectively.Other markers of risk include mutations in the presenilin genes, PS1 andPS2, and ApoE4 gene, a family history of AD, and hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia by the presenceof risk factors described above. In addition, a number of diagnostictests are available for identifying individuals who have AD. Theseinclude measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by Alzheimer's Diseaseand Related Disorders Association criteria.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30 years of age). Usually, however, it is not necessary to begintreatment until a patient reaches 40, 50, 60, or 70 years of age.Treatment typically entails multiple dosages over a period of time.Treatment can be monitored by assaying antibody, or activated T-cell orB-cell responses to the therapeutic agent over time. If the responsefalls, a booster dosage is indicated. In the case of potential Down'ssyndrome patients, treatment can begin antenatally by administeringtherapeutic agent to the mother or shortly after birth.

In prophylactic applications, pharmaceutical compositions containing theimmunogenic tau peptides are administered to a patient susceptible to,or otherwise at risk of, Alzheimer's disease or other tauopathy in anamount sufficient to eliminate or reduce the risk, lessen the severity,or delay the outset of the disease, including biochemical, histologicand/or behavioral symptoms of the disease, its complications andintermediate pathological phenotypes presented during development of thedisease. In therapeutic applications, compositions containing a tauantibody are administered to a patient suspected of, or alreadysuffering from, such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease. Insome methods, administration of agent reduces or eliminates mildcognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. Typically, the immuneresponse is monitored and repeated dosages are given if the immuneresponse starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including mode of administration, target site,physiological state of the patient, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. The amount of immunogendepends on whether adjuvant is also administered, with higher dosagesbeing required in the absence of adjuvant. The amount of an immunogenfor administration sometimes varies from 1-500 μg per patient and moreusually from 5-500 μg per injection for human administration.Occasionally, a higher dose of 1-2 mg per injection is used. Typicallyabout 10, 20, 50, or 100 μg is used for each human injection. The massof immunogen also depends on the mass ratio of immunogenic epitopewithin the immunogen to the mass of immunogen as a whole. Typically,10⁻³ to 10⁻⁵ micromoles of immunogenic epitope are used for eachmicrogram of immunogen. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. On any given day thata dosage of immunogen is given, the dosage is greater than 1 μg/patientand usually greater than 10 μg/patient if adjuvant is also administered,and greater than 10 μg/patient and usually greater than 100 μg/patientin the absence of adjuvant. A typical regimen consists of animmunization followed by booster injections at time intervals, such as 6week intervals. Another regimen consists of an immunization followed bybooster injections 1, 2, and 12 months later. Another regimen entails aninjection every two months for life. Alternatively, booster injectionscan be on an irregular basis as indicated by monitoring of immuneresponse.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per every two weeks or once a month or onceevery 3 to 6 months. In some methods, two or more monoclonal antibodieswith different binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated. Antibody is usually administered on multipleoccasions. Intervals between single dosages can be weekly, monthly, oryearly. In some methods, dosage is adjusted to achieve a plasma antibodyconcentration of 1-1000 μg/ml and in some methods 25-300 μg/ml.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, human antibodies show the longest half life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies. The dosage and frequency of administration can varydepending on whether the treatment is prophylactic or therapeutic. Inprophylactic applications, a relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage at relatively shortintervals is sometimes required until progression of the disease isreduced or terminated, and preferably until the patient shows partial orcomplete amelioration of symptoms of disease. Thereafter, the patent canbe administered a prophylactic regime.

Doses for nucleic acids encoding immunogens range from about 10 ng toabout 1 g, from about 100 ng to about 100 mg, from about 1 μg to about10 mg, or from about 30 to about 300 μg DNA per patient. Doses forinfectious viral vectors vary from 10-100, or more, virions per dose.

Agents for inducing an immune response can be administered byparenteral, topical, intravenous, oral, subcutaneous, intraarterial,intracranial, intraperitoneal, intranasal, or intramuscular means forprophylactic and/or therapeutic treatment. The most typical route ofadministration of an immunogenic agent is subcutaneous, although otherroutes can be equally effective. The next most common route isintramuscular injection. This type of injection is most typicallyperformed in the arm or leg muscles. In some cases, it may be desirableto inject the therapeutic agent of the present invention directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionis preferred for administration of antibody. In some methods, particulartherapeutic antibodies are injected directly into the cranium. In somemethods, antibodies are administered as a sustained release compositionor device, such as a Medipad™ device (Elan Pharm. Technologies, Dublin,Ireland).

Another aspect of the present invention is directed to a combinationtherapy where an immunogenic tau peptide or antibody recognizing animmunogenic tau epitope of the present invention is administered incombination with agents that are effective for the prevention ortreatment of other conditions or diseases associated with, or resultingfrom, the deposition of amyloidogenic proteins or peptides.Amyloidogenic proteins/peptides subject to deposition include, withoutlimitation, beta protein precursor, prion and prion proteins,α-synuclein, tau, ABri precursor protein, ADan precursor protein, isletamyloid polypeptide, apolipoprotein AI, apolipoprotein AII, lyzozyme,cystatin C, gelsolin, atrial natriuretic factor, calcitonin,keratoepithelin, lactoferrin, immunoglobulin light chains,transthyretin, A amyloidosis, β2-microglobulin, immunoglobulin heavychains, fibrinogen alpha chains, prolactin, keratin, and medin.Therefore, a combination therapeutic of the present invention wouldinclude an immunogenic tau peptide or antibody recognizing animmunogenic tau epitope and an agent or agents targeting one or more ofthe aforementioned amyloidogenic proteins or peptides.

In the case of amyloidogenic diseases such as, Alzheimer's disease andDown's syndrome, immune modulation to clear amyloid-beta (Aβ) depositsis an emerging therapy. Immunotherapies targeting Aβ have consistentlyresulted in cognitive improvements. It is likely that tau and Aβpathologies are synergistic. Therefore, combination therapy targetingthe clearance of both tau and Aβ and Aβ-related pathologies at the sametime may be more effective than targeting each individually.

In the case of Parkinson's Disease and related neurodegenerativediseases, immune modulation to clear aggregated forms of the α-synucleinprotein is also an emerging therapy. A combination therapy which targetsthe clearance of both tau and α-synuclein proteins simultaneously may bemore effective than targeting either protein individually.

In the case of prion disease and related neurodegenerative diseases,immune modulation to clear the disease associated form of the prionprotein, PrP^(Sc), is an emerging therapy. Therefore, a combinationtherapy which targets the clearance of both tau and the pathologicalPrP^(Sc) protein simultaneously may be more effective than targetingeither protein individually.

Individuals with type-2 diabetes may be more prone to the development ofAlzheimer's disease. Therefore, a combination therapy which includes anagent targeting the clearance of islet amyloid polypeptide and an agentpreventing the development or progression of Alzheimer's diseases (i.e.,preventing tau deposition) would have enhanced therapeutic benefit tothe individual.

Another aspect of the present invention relates to a method ofdiagnosing an Alzheimer's disease or other tauopathy in a subject. Thismethod involves detecting, in the subject, the presence of pathologicaltau conformer using a diagnostic reagent, where the diagnostic reagentis an antibody, or active binding fragment thereof, of the presentinvention. As described supra, the antibody has antigenic specificityfor an isolated tau peptide having an amino acid sequence selected fromSEQ ID NOs: 2-75 or SEQ ID NOs: 101-103. The diagnosis of theAlzheimer's disease or other tauopathy is based on the detection of apathological tau conformer in the subject.

Detecting the presence of a pathological tau conformer in a subjectusing the diagnostic antibody reagent of the present invention can beachieved by obtaining a biological sample from the subject (e.g., blood,urine, cerebral spinal fluid), contacting the biological sample with thediagnostic antibody reagent, and detecting binding of the diagnosticantibody reagent to a pathological tau protein conformer in the samplefrom the subject. Assays for carrying out the detection of apathological tau protein in a biological sample using the diagnosticantibody of the present invention are well known in the art and include,without limitation, ELISA, immunohistochemistry, western blot.

Alternatively, detecting the presence of a pathological tau proteinconformer in a subject using the diagnostic antibody reagent of thepresent invention can be achieved using in vivo imaging techniques. Invivo imaging involves administering to the subject the diagnosticantibody having antigenic specificity for a pathological tau peptide orepitope (i.e., SEQ ID NOs: 2-75 and 101-103) and detecting binding ofthe diagnostic antibody reagent to the pathological tau proteinconformer in vivo. As described supra, preferred antibodies bind to thepathological tan protein without binding to non-tau proteins and withoutbinding to the non-pathological forms of tau.

Diagnostic antibodies or similar reagents can be administered byintravenous injection into the body of the patient, or directly into thebrain by intracranial injection. The dosage of antibody should be withinthe same ranges as for treatment methods. Typically, the antibody islabeled, although in some methods, the primary antibody with affinityfor the pathological tau protein is unlabelled and a secondary labelingagent is used to bind to the primary antibody. The choice of labeldepends on the means of detection. For example, a fluorescent label issuitable for optical detection. Use of paramagnetic labels is suitablefor tomographic detection without surgical intervention. Radioactivelabels can also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled pathological tau conformers, tau aggregates, and/orneurofibrillary tangles in a sample from the subject or in the subject,to corresponding baseline values. The base line values can represent themean levels in a population of undiseased individuals. Baseline valuescan also represent previous levels determined in the same subject.

The diagnostic methods described above can also be used to monitor asubject's response to therapy. In this embodiment, detecting thepresence of pathological tau in a subject is determined prior to thecommencement of treatment. The level of pathological tau in the subjectat this timepoint is used as a baseline value. At various times duringthe course of treatment the detection of pathological tau proteinconformers, tau aggregates, and/or neurofibrillary tangles is repeated,and the measured values thereafter compared with the baseline values. Adecrease in values relative to baseline signals a positive response totreatment. Values can also increase temporarily in biological fluids aspathological tau is being cleared from the brain.

The present invention is further directed to a kit for performing theabove described diagnostic and monitoring methods. Typically, such kitscontain a diagnostic reagent, preferably the antibody of the presentinvention that has antigenic specificity for a pathological tau peptide(i.e., SEQ ID NOs: 2-75 and 101-103). The kit can also include adetectable label. The diagnostic antibody itself may contain thedetectable label (e.g., fluorescent molecule, biotin, etc.) which isdirectly detectable or detectable via a secondary reaction (e.g.,reaction with streptavidin). Alternatively, a second reagent containingthe detectable label may be utilized, where the second reagent hasbinding specificity for the primary antibody. In a diagnostic kitsuitable for measuring pathological tau protein in a biological sample,the antibodies of the kit may be supplied prebound to a solid phase,such as to the wells of a microtiter dish.

Diagnostic kits of the present invention also include kits that areuseful for detecting antibody production in a subject followingadministration of an immunogenic tau peptide of the present invention.Typically, such kits include a reagent that contains the antigenicepitope of the antibodies generated by the subject. The kit alsoincludes a detectable label. In a preferred embodiment, the label istypically in the form of labeled anti-idiotypic antibodies. The reagentof the kit can be supplied prebound to a solid phase, such as to thewells of a microtiter dish.

The following examples illustrate various methods for compositions inthe treatment method of the invention. The examples are intended toillustrate, but in no way limit, the scope of the invention.

EXAMPLES Example 1 Peptides

The peptide immunogens were synthesized at the Keck facility (YaleUniversity), by the solid-phase technique on a p-methyl-benzhydrylamineresin, using a Biosearch SAM 2 synthesizer (Biosearch, Inc., San Rafael,Ca.). The peptides were cleaved from the resin with HF and thenextracted with ether and acetic acid before lyophilization.Subsequently, the peptides were purified by HPLC with the use of areverse-phase support medium (Delta-Bondapak) on a 0.78×30 cm columnwith a 0-66% linear gradient of acetonitrile in 0.1% TFA.

Example 2 Animals Used in Studies

Studies were performed in the transgenic (Tg) JNPL3 P301L mouse modelthat develops neurofibrillary tangles in several brain regions andspinal cord (Taconic, Germantown, N.Y.) (Lewis et al., “NeurofibrillaryTangles, Amyotrophy and Progressive Motor Disturbance in Mice ExpressingMutant (P301L) Tau Protein,” Nat Genet. 25:402-405 (2000), which ishereby incorporated by reference in its entirety). While this model isnot ideal for AD, it is an excellent model to study the consequences oftangle development and for screening therapy that may prevent thegeneration of these aggregates. Another advantage of these animals isthe relatively early onset of pathology. In the homozygous line,behavioral abnormalities associated with tau pathology can be observedat least as early as 3 months, but the animals remain relatively healthyat least until 8 months of age. In other words, at 8 months, the animalsambulate, feed themselves, and can perform the behavioral taskssufficiently well to allow the treatment effect to be monitored.

In addition to the JNPL3 P301L model, studies were also carried outusing an htau/PS1 (M146L) mouse model (Boutajangout et al., “Presenilin1 Mutation Promotes Tau Phosphorylation and Aggregation in a NovelAlzheimer's Disease Mouse Model,” Alzheimer's and Dementia 4:T185(2008), which is hereby incorporated by reference in its entirety). htaumice express unmutated human tau protein on a null mouse tau backgroundand better resembles Alzheimer's tau pathology in the age of onset andbrain distribution (Andorfer et al., “Hyperphosphorylation andAggregation of Tau in Mice Expressing Normal Human Tau Isoforms,” JNeurochem 86: 582-90 (2003), which is hereby incorporated by referencein its entirety). The PS1 model, carrying a mutation (M146L) in thePresenlin 1 protein, has shown to have increased Aβ levels and topromote Aβ deposition when crossed with Tg2576 mice (Duff et al.,“Increased Amyloid-beta 42(43) in Brains of Mice Expressing MutantPresenilin 1,” Nature 383:710-713 (1996) and Holcomb et al.,“Accelerated Alzheimer-Type Phenotype in Transgenic Mice Carrying BothMutant Amyloid Precursor Protein and Presenilin 1 Transgenes,” NatureMed 4:97-100 (1998), which are hereby incorporated by reference in theirentirety).

htau mice, expressing all six human isoforms of tau, were crossed withPS1 (M146L) mice and maintained on a mouse tau knockout background(htau/PS1/mtau−/−). The PS1 mutation promotes hyperphosphorylation oftau in this model which leads to more aggressive tau pathology withearlier onset than in the htau model (Boutajangout et al., “Presenilin 1Mutation Promotes Tau Phosphorylation and Aggregation in a NovelAlzheimer's Disease Mouse Model,” Alzheimer's and Dementia 4:T185(2008), which is hereby incorporated by reference in its entirety).

Example 3 Vaccine Administration

Phos-tau peptides were mixed with Adju-Phos adjuvant (BrenntagBiosector, Denmark) at a concentration of 1 mg/ml and the solution wasrotated overnight at 4° C. prior to administration to allow the peptideto adsorb onto the aluminum phosphate particles.

JNPL3 P301L mice received a subcutaneous injection of 100 μl followed bya second injection 2 weeks later and then monthly thereafter (unlessotherwise indicated). Vaccination started at 2-3 months of age andcontinued until the animals were 8-9 months of age at which time theanimals were perfused and their organs collected for analysis. The micewent through a battery of sensorimotor tests at 5-6 months and again at8-9 months of age prior to sacrifice. Control mice received the adjuvantalone.

htau/PS1/mtau−/− mice (n=12) were immunized with the phosphorylated tauimmunogen Tau379-408[P-Ser_(396,404)]. Three non-immunized controlgroups were included that received adjuvant alone. The main controlgroup consisted of identical mice that were not immunized (htau/PS1controls; n=16). Other control groups were htau/PS1 mice that expressedmouse tau (htau/PS1/mtau; n=8) as well as htau littermates on a mousetau knockout background (htau controls; n=10).

htau/PS1/mtau−/− mice (3-4 months of age) received 100 μg of thephosphorylated tau derivative intraperitoneally (i.p.) in alum adjuvantwith the first 3 injections every 2 weeks. Subsequent administration wasat monthly intervals. The control groups received adjuvant alone. At 7-8months the mice went through extensive behavioral testing to determinetreatment efficacy, and were subsequently killed for analysis at 8-9months of age. Locomotor activity, traverse beam, and rotarod tests wereperformed to determine if measured cognitive deficits in the learningand memory tasks could be attributed to sensorimotor abnormalities.Cognitive testing was performed using the radial arm maze, the closedfield symmetrical maze, and the object recognition test (Sigurdsson etal., “An Attenuated Immune Response is Sufficient to Enhance Cognitionin an Alzheimer's Disease Mouse Model Immunized with Amyloid-betaDerivatives,” J Neurosci 24:6277-6282 (2004), Asuni et al., “Vaccinationof Alzheimer's Model Mice with Abeta Derivative in Alum Adjuvant ReducesAbeta Burden Without Microhemorrhages.” Eur J Neurosci. 24:2530-42(2006), and Asuni et al., “Immunotherapy Targeting Pathological TauConformers in a Tangle Mouse Model Reduces Brain Pathology withAssociated Functional Improvements,” J Neurosci 27:9115-9129 (2007),which are hereby incorporated by reference in their entirety).

Example 4 Tau Immunotherapy Generates a Robust Antibody Response

The mice were bled prior to the commencement of the study (T0), a weekfollowing the third injection, periodically thereafter, and at sacrifice(Tf). The antibody response to the vaccine was determined by dilution ofplasma (1:200 unless otherwise indicated) using an ELISA assay asdescribed previously (Sigurdsson et al., “Immunization with aNon-Toxic/Non-Fibrillar Amyloid-β Homologous Peptide Reduces Alzheimer'sDisease Associated Pathology in Transgenic Mice,” Am J Pathol.159:439-447 (2001) and Sigurdsson et al., “An Attenuated Immune Responseis Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse ModelImmunized with Amyloid-beta Derivatives,” J. Neurosci. 24:6277-6282(2004), which are hereby incorporated by reference in their entirety),where the immunogen was coated onto Immulon™ microtiter wells (ThermoFischer Scientific. Waltham, Mass.). For detection, goat anti-mouse IgG(Pierce, Rockford, Ill.) or anti-mouse IgM (Sigma, St. Louis, Mo.)linked to a horseradish peroxidase were used at 1:3000 dilution.Tetramethyl benzidine (Pierce) was the substrate.

FIG. 1A shows the robust IgG and IgM immune response in JNPL3 P301Ltangle mice immunized with Tau210-216[P-Thr₂₁₂-Ser₂₁₄] (SEQ ID NO: 2)linked to tetanus toxin helper T-cell epitope (TT947-967) via GPSLlinker. Mice of 2-3 months of age received the first two immunizationstwo weeks apart and then monthly thereafter. To assess antibodyresponse, the mice were bled prior to the first immunization,periodically thereafter one week after vaccine administration, and whenthe mice were killed for tissue harvesting at 8-9 months of age. FIG. 1Ashows IgG and IgM antibody response measured one week after the 6^(th)immunization (T3) and again at 8-9 months of age, which was at the timeof sacrifice (Tf=Tfinal). FIG. 1B shows that a strong antibody responsewas generated against the tetanus toxin epitope itself as assessed byIgG and IgM binding to an unrelated tau epitope Tau260-264[P-Ser₂₆₂]linked via GPSL to TT947-967.

JNPL3 P301L tangle mice immunized with Tau260-264[P-Ser₂₆₂] (SEQ IDNO:3) linked to tetanus toxin helper T-cell epitope (TT947-967) via GPSLlinker generated a robust IgG response against the immunogen at shown inFIG. 2A. As above, the mice received the first two immunizations twoweeks apart and then monthly thereafter from 2-3 months of age until 8-9months of age. A good portion of that antibody response is generatedagainst the tetanus toxin epitope as assessed by IgG binding to anunrelated tau epitope Tau210-216[P-Thr₂₁₂-Ser₂₁₄] linked via GPSL toTT947-967 (FIG. 23). However, as shown in FIG. 2C, a good portion of theantibody response is also generated against the tau epitope as assessedby IgG binding to a larger tau epitope Tau240-270[P-Ser₂₆₂] thatcontains the Tau260-264[P-Ser₂₆₂] region. T0-Tfinal: Bleed prior tovaccination (T0), one week after third -(T1), sixth -(T2), seventh (T3)immunization, and at tissue harvesting (Tf).

A robust antibody (IgG) response was generated in JNPL3 P301L tanglemodel mice immunized in with Tau229-237[P-Thr₂₃₁-Ser₂₃₅] (SEQ ID NO: 4)linked to tetanus toxin helper T-cell epitope (TT947-967] (FIG. 3). Themice were immunized from 2-3 months of age, two weeks apart and a monthlater, and bled (T1) one week after the third immunization.

A robust antibody (IgG) response was also generated in JNPL3 P301Ltangle model mice immunized with the pseudophosphorylated immunogen,Tau379-408[Asp_(396, 404)] (SEQ ID NO: 57) in alum adjuvant.Importantly, these antibodies recognize the phospho-epitope.Tau379-408[P-Ser_(396, 404)], to a similar degree. The mice wereimmunized from 2-3 months of age, every two weeks for the first twoimmunizations, and monthly thereafter. The mice were bled (Tf=Tfinal) atthe time of tissue harvesting at 7-8 months of age.

Example 5 Tau Immunotherapy Reduces Tau Aggregation in the Brain

For histological analysis of tau pathology, mice were anesthetized withsodium pentobarbital (120 mg/kg, i.p.), perfused transaortically withPBS and the brains processed as previously described (Sigurdsson et al.,“Immunization with a Non-Toxic/Non-Fibrillar Amyloid-β HomologousPeptide Reduces Alzheimer's Disease Associated Pathology in TransgenicMice,” Am J Pathol 159:439-447 (2001): Sigurdsson et al., “An AttenuatedImmune Response is Sufficient to Enhance Cognition in an Alzheimer'sDisease Mouse Model Immunized with Amyloid-beta Derivatives,”J Neurosci24:6277-6282 (2004); and Sigurdsson E., “Histological Staining ofAmyloid-beta in Mouse Brains,” Methods Mol Biol 299:299-308 (2005),which are hereby incorporated by reference in their entirety). Briefly,the right hemisphere was immersion fixed overnight inperiodate-lysine-paraformaldehyde (PLP), whereas the left hemisphere wassnap-frozen for tau protein analysis. Following fixation, the brain wasmoved to a phosphate buffer solution containing 20% glycerol and 2%dimethylsulfoxide (DMSO) and stored at 4° C. until sectioned. Serialcoronal brain sections (40 μm) were cut and every tenth section wasstained with the PHF1 monoclonal antibody that recognizes phosphorylatedserines 396 and 404 located within the microtubule-binding repeat on theC-terminal of PHF tau protein (Otvos et al., “Monoclonal Antibody PHF-1Recognizes Tau Protein Phosphorylated at Serine Residues 396 and 404,” JNeurosci Res 39:669-673 (1994), which is hereby incorporated byreference in its entirety)

Tau antibody staining was performed as described in Sigurdsson et al.,“Immunization with a Non-Toxic/Non-Fibrillar Amyloid-β HomologousPeptide Reduces Alzheimer's Disease Associated Pathology in TransgenicMice,” Am J Pathol 159:439-447 (2001) and Sigurdsson et al., “AnAttenuated Immune Response is Sufficient to Enhance Cognition in anAlzheimer's Disease Mouse Model Immunized with Amyloid-betaDerivatives,” J Neurosci 24:6277-6282 (2004), which are herebyincorporated by reference in their entirety. Briefly, sections wereincubated in the primary PHF1 antibody at a 1:100 to 1:1000 dilution. Amouse on mouse immunodetection kit (Vector Laboratories, Burlingame,Calif.) was used, in which the anti-mouse IgG secondary antibody wasused at a 1:2000 dilution.

Analysis of tissue sections was quantified with a Bioquant imageanalysis system. The software uses hue, saturation, and intensity tosegment objects in the image field. Thresholds were established withaccurately identified objects on a standard set of slides and thesesegmentation thresholds remained constant throughout the analysissession. After establishing the threshold parameter, the image field wasdigitized with a frame grabber. The Bioquant software corrects forheterogeneity in background illumination (blank field correction) andcalculates the measurement parameter for the entire field. Forquantitative image analysis of immunohistochemistry, the granular layerof the dentate gyrus was initially selected which consistently containedintraneuronal tau aggregates (pretangles and tangles). This observationconcurs with the original characterization of this model (Lewis et al.,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet. 25:402-405(2000), which is hereby incorporated by reference in its entirety). Allprocedures were performed by an individual blind to the experimentalconditions of the study. Sample numbers were randomized before the startof the tissue processing, and the code was broken only after theanalysis was complete. Every tenth section from the mouse brain wassampled and the measurement was the percent of area in the measurementfield at X200 magnification occupied by reaction product with the tip ofthe dentate gyrus at the left edge of the field. Four to five sectionswere analyzed per animal.

Immunization of homozygous JNPL3 tau P301L mice withTau260-264[P-Ser₂₆₂] (SEQ ID NO: 3) linked to TT947-967 (T299) reducedthe levels of pathological tau in both the brain stem (FIG. 5A) and thedentate gyrus (FIG. 5B) compared to control mice receiving adjuvantonly. Similarly, immunization of htau/PS1 mice with the phosphorylatedTau379-408[P-Ser_(396,404)] immunogenic peptide reduced the amount oftau aggregates by 56% in the pyriform cortex (FIG. 6, compare htau/PS1immunized vs. htau/PS1 controls). Significant difference was observedbetween the immunized and control groups (one-way ANOVA, p<0.01). Posthoc analysis also showed that immunized htau/PS1 mice differed fromtheir htau/PS1 controls (p<0.01). ** p<0.01.

Example 6 Tau Immunotherapy Prevents Cognitive Decline

To determine if the tau immunotherapy prevented or reversed theage-related sensorimotor abnormalities observed in the P301L or if itcaused any motor impairments in the htau/PS1 mice, animals administeredthe immunogenic Tau 260-264[P-Ser₂₆₂] (SEQ ID NO: 3) or Tau379-408[P-Ser_(396, 404)] (SEQ ID NO: 82) were assessed using a varietyof sensorimotor and cognitive tests described below.

Rotarod Test:

Animals were placed onto the rod (diameter 3.6 cm) apparatus to assessdifferences in motor coordination and balance by measuring fore- andhindlimb motor coordination and balance (Rotarod 7650 acceleratingmodel; Ugo Basile, Biological Research Apparatus, Varese, Italy). Thisprocedure was designed to assess motor behavior without a practiceconfound. The animals were habituated to the apparatus by receivingtraining sessions of two trials, sufficient to reach a baseline level ofperformance. Then, the mice were tested three additional times, withincreasing speed. During habituation, the rotarod was set at 1.0 rpm,which was gradually raised every 30 sec, and was also wiped clean with30% ethanol solution after each session. A soft foam cushion was placedbeneath the apparatus to prevent potential injury from falling. Eachanimal was tested for three sessions (data combined for subsequentanalysis), with each session separated by 15 min, and measures weretaken for latency to fall or invert (by clinging) from the top of therotating barrel.

Traverse Beam:

This task tests balance and general motor coordination and functionintegration. Mice were assessed by measuring their ability to traverse agraded narrow wooden beam to reach a goal box (Torres et al.,“Behavioral, Histochemical and Biochemical Consequences of SelectiveImmunolesions in Discrete Regions of the Basal Forebrain CholinergicSystem,” Neuroscience 63:95-122 (1994), which is hereby incorporated byreference in its entirety). The mice were placed on a 1.1 cm wide beamthat is 50.8 cm long and suspended 30 cm above a padded surface by twoidentical columns. Attached at each end of the beam is a shaded goalbox. Mice were placed on the beam in a perpendicular orientation tohabituate and were then monitored for a maximum of 60 sec. The number offoot slips each mouse had before falling or reaching the goal box wererecorded for each of four successive trials. Errors are defined asfootslips and were recorded numerically.

Radial Arm Maze:

The maze apparatus is an 8-arm elevated radial maze constructed fromPlexiglas. Each arm is 35 cm long and 7 cm wide with a water cup 1 cm indiameter positioned at the end of each arm. Sidewalls 15 cm high extend12 cm into each arm to prevent animals from crossing between arms. Thecentral area is an octagonal shaped hub 14 cm in diameter. ClearPlexiglas guillotine doors, operated remotely by a pulley system controlaccess to the arms. The maze is elevated 75 cm above floor level andsituated in a room in which several distinctive objects of a constantlocation serve as extra maze cues. Prior to testing, mice were adaptedfor 5 days. During this period, the mice received 0.1% saccharine inwater for 1 hour per day and were then adapted 16 hours later to accessthe sugar solution from a cup placed at the end of each arm. The firsttwo days of adaptation were performed in a Y-maze which the mice wereallowed to explore freely. The subsequent three days of adaptation wereperformed in the radial arm maze, in which the doors were raised andlowered periodically to accustom the animals to the sound associatedwith their operation. The same water deprivation schedule was maintainedduring the 9 day testing period. The mice maintain good health on thisschedule. Each testing trial was begun by placing the mouse in thecentral area and raising all doors. When an arm was entered all doorswere lowered. After the mouse consumed the saccharine water, the door tothat arm was raised allowing the mouse to return to the central arena.After a 5 sec interval, the next trial was initiated by again raisingall of the doors simultaneously. This procedure was continued until theanimal had entered all 8 arms or until 10 min has elapsed. Dailyacquisition sessions were continued for 9 days. The number of errors(entries to previously visited arms) and time to complete each sessionwere recorded.

Object Recognition:

The spontaneous object recognition test that was utilized measuresdeficits in short term memory, and was conducted in a square-shapedopen-field box (48 cm square, with 18 cm high walls constructed fromblack Plexiglas), raised 50 cm from the floor. The light intensity wasset to 30 lx. On the day before the tests, mice were individuallyhabituated in a session in which they were allowed to explore the emptybox for 15 min. During training sessions, two novel objects were placedat diagonal corners in the open field and the animal was allowed toexplore for 15 min. For any given trial, the objects in a pair were 10cm high, and composed of the same material so that they could notreadily be distinguished by olfactory cues. The time spent exploringeach object was recorded by a tracking system (San Diego Instruments,San Diego, Calif.), and at the end of the training phase, the mouse wasremoved from the box for the duration of the retention delay (RD=3 h).Normal mice remember a specific object after a delay of up to 1 h andspend the majority of their time investigating the novel object duringthe retention trial. During retention tests, the animals were placedback into the same box, in which one of the previous familiar objectsused during training was replaced by a second novel object, and allowedto explore freely for 6 min. A different object pair was used for eachtrial for a given animal, and the order of exposure to object pairs aswell as the designated sample and novel objects for each pair werecounterbalanced within and across groups. The time spent exploring thenovel and familiar objects was recorded for the 6 min.

Closed Field Symmetrical Maze:

This apparatus is a rectangular field 30 cm square with 9 cm high wallsdivided into 36, 9.5 cm squares and covered by a clear Plexiglas top.Endboxes, each 11×16×9 cm, are situated at diagonal corners of thefield. The symmetrical maze is a modification of the Hebb-Williams andRabinovitch-Rosvold type of tests, as discussed previously (Asuni etal., “Vaccination of Alzheimer's Model Mice with Abeta Derivative inAlum Adjuvant Reduces Abeta Burden without Microhemorrhages,” Eur JNeurosci 24:2530-2542 (2006), which is hereby incorporated by referencein its entirety). Briefly, the main difference is that eachend-compartment functions as both a startbox and a goalbox, and the micerun in opposite direction on alternate trials, thereby eliminatingintertrial handling. The barriers are placed in the field in symmetricalpatterns, so that mice face the same turns going in either directionwithin a given problem. Prior to testing, the mice were adapted to awater restriction schedule (2 h daily access to water). The mice weregiven two adaptation sessions prior to the beginning of testing. In thefirst session, all animals were given saccharine flavored water in thegoal box for 10 min. In session 2, they were placed in the start chamberand permitted to explore the field and enter the goal box where waterreward (0.05 mL) was available. When the mice were running reliably fromthe start chamber to the goal box, they were given three practicesessions on simple problems where one or two barriers were placed indifferent positions in the field so as to obstruct direct access to thegoal box. Formal testing consisted of the presentation of three problemsgraded in difficulty based on previous data (Asuni et al., “Vaccinationof Alzheimer's Model Mice with Abeta Derivative in Alum Adjuvant ReducesAbeta Burden without Microhemorrhages,” Eur J Neurosci 24:2530-2542(2006), which is hereby incorporated by reference in its entirety) andpublished norms for mice. One problem was presented per day and the micewere given five trials on each problem with an intertrial interval of 2min. Performance was scored manually by the same observer in terms oferrors (i.e., entries and reentries into designated error zones) andtime to complete each trial.

The objective of these experiments was to evaluate the effects of thevaccination on selected sensorimotor (i.e., traverse beam and rotarod)and cognitive behaviors (i.e., radial arm maze, object recognition test,and closed field symmetrical maze test). The homozygous P301L mice havetangle pathology as early as 3 months of age and those animals weretested at 5 and 8 months of age. The htau/PS1 animals were tested at 7-8months of age.

Immunization of homozygous JNPL3 tau P301L mice with the phosphorylatedimmunogenic tau peptide Tau260-264[P-Ser₂₆₂] linked to the tetanus toxinhelper T-cell epitope (TT947-967) prevented functional impairmentassociated with the development of neurofibrillary tangles as assessedusing the traverse beam test at 8 months of age (FIG. 7A) and therotarod test at 5-6 months of age and at 8-9 months of age (FIG. 7B).Control JNPL3 tau P301L mice received adjuvant alone.

Immunization of htau/PS1 mice with the phosphorylatedTau379-408[P-Ser396,404] prevented cognitive decline in all three teststhat were employed: 1) the radial arm maze (RAM: two-way ANOVA repeatedmeasures, p<0.0001, FIG. 8A), 2) the object recognition test (ORT;one-way ANOVA, p=0.005, FIG. 8B), and 3) the closed field symmetricalmaze (CFSM; one-way ANOVA, Maze A: p<0.001, Maze B: p<0.0001, Maze C:p<0.01, FIGS. 9A-9C). In the RAM and the CFSM, the immunized htau/PS1mice performed better than the control htau/PS1 mice on all the days(RAM; p<0.01-0.001) and in all the mazes that were of increasingcomplexity, as indicated by the number of errors (note that the Y axisscale differs; CFSM Maze A: p<0.01, Mazes B, C: p<0.001). In the ORT,post hoc analysis revealed that the immunized htau/PS1 mice had bettershort-term memory than identical control mice (p<0.01). It is wellestablished that cognitively normal mice spend about 70% of their timewith the new object compared to the old object (Asuni et al.,“Immunotherapy Targeting Pathological Tau Conformers in a Tangle MouseModel Reduces Brain Pathology with Associated Functional Improvements,”J Neurosci 27:9115-129 (2007), which is hereby incorporated by referencein its entirety). The immunized htau/PS1 mice did not differsignificantly from their non-immunized identical control mice in any ofthe sensorimotor tasks (rotarod, traverse beam, locomotor activity).These findings indicate that the cognitive improvements observedfollowing the immunization cannot be explained by sensorimotor effects,which further strengthens the results.

Example 7 Tau Immunotherapy Reduces Levels of Pathological Tau

Brain tissue was homogenized in a buffer containing 0.1 mM2-(N-morpholino) ethanosulfonic acid, 0.5 mM MgSO₄, 1 mM EGTA, 2 mMdithiothreitol, pH 6.8, 0.75 mM NaCl, 2 mM phenylmethyl sulfonylfluoride, Complete mini protease inhibitor mixture (1 tablet in 10 ml ofwater; Roche) and phosphatase inhibitors (20 mM NaF and 0.5 mM sodiumorthovanadate). The homogenate was then centrifuged (20,000×g) for 30min at 4° C. to separate a soluble cytosolic fraction (supernatant 1)and insoluble fraction (pellet 1). The pellet was resuspended in thesame volume of buffer without protease and phosphatase inhibitors, butthat contained 1% (v/v) Triton X-100 and 0.25% (w/v) desoxycholatesodium and ultracentrifuged at 50,000 for 30 min to obtain adetergent-extracted supernatant 2 that was analyzed as insolublefraction. Supernatant 1 and 2 were heated at 100° C. for 5 min and thesame amount of protein was electrophoresed on 12% (w/v) polyacrylamidegel. The blots were blocked in 5% non-fat milk with 0.1% Tween-20 inTBS, and incubated with different antibodies overnight, and then washedand incubated at room temperature for 1 h with peroxidase-conjugated,anti-mouse or anti-rabbit IgG. Subsequently, the bound antibodies weredetected by ECL (Pierce). Densitometric analysis of immunoblots wereperformed by NIH Image J program and the levels of pathological tan wasnormalized relative to the amounts of total tau protein instead of actinlevels, as some studies have reported that changes in pathophysiologicalconditions and interactions with extracellular matrix components canalter actin protein synthesis, rendering actin unsuitable as an internalstandard.

For Western blot analysis, total tau was measured with polyclonal B19antibody whereas pathological tau was detected with monoclonal PHF1antibody (FIGS. 10A-10F). Levels of total soluble and insoluble tau didnot differ significantly between the groups (FIGS. 10A-10B), whereaslevels of soluble PHF1 stained tau were significantly decreased (41%,p<0.001) in the immunized mice compared to their identical controls(FIG. 10C). A trend was observed for a decrease (22%) in insoluble PHF1reactive tau (FIG. 10D). Further analysis indicated a very strong trendfor the immunotherapy to reduce the ratio of PHF1/B19 by 35% and 43% inthe soluble and insoluble fractions, respectively (FIGS. 10E and 10F).These findings indicate that pathological tau was preferentially beingcleared.

Importantly, cognitive improvements observed in the htau/PS1 micereceiving immunotherapy correlated well with reduction in PHF1 stainedtau aggregates assessed by immunohistochemistry. Significant correlationwas observed in all three memory tests (RAM (last day of testinganalyzed): r=0.36, p=0.01; CFSM: Maze A, r=0.33, p=0.02; Maze C, r=0.40,p=0.01; ORT: r=−0.31, p=0.03). With regard to the western blotfractions, significant correlation was observed in both soluble andinsoluble fractions and their ratios relative to total tau in the radialarm maze (soluble PHF1: r=0.41, p<0.01; soluble PHF1/total soluble tau:r=0.34, p<0.05; insoluble PHF1: r=0.52, p<0.001; insoluble PHF1/totalinsoluble tau: r=0.33, p<0.05) but not in the two other cognitive tests.

Example 8 Passive immunotherapy Targeting the P-396, 404 EpitopePrevents Functional Decline and Reduces Tau Aggregates in the Brain

To determine the feasibility of passive immunotherapy, homozygous P301Lmice were injected intraperitoneally (i.p.) with PHF1, a monoclonal tauantibody (provided by Dr. Peter Davies) that recognizes NFT andpretangles in the P301L (JNPL3) mouse model and in AD (Lewis et al,“Neurofibrillary Tangles, Amyotrophy and Progressive Motor Disturbancein Mice Expressing Mutant (P301L) Tau Protein,” Nat Genet. 25:402-40522(2000), which is hereby incorporated by reference in its entirety). Thismonoclonal antibody recognizes tau that is phosphorylated on serineamino acids 404 and 396 on the C-terminal of tau (Greenberg et al.,“Hydrofluoric Acid-Treated Tau PHF Proteins Display the Same BiochemicalProperties as Normal Tau,” J Biol Chem 267:564-569 (1992) which ishereby incorporated by reference in its entirety). Therefore, it is amonoclonal analog of the prototype of one active immunization approach(Asuni et al., “Immunotherapy Targeting Pathological Tau Conformers in aTangle Mouse Model Reduces Brain Pathology with Associated FunctionalImprovements,” J Neurosci 27:9115-9129 (2007), which is herebyincorporated by reference in its entirety), Tau379-408[P-Ser396,404]that contains the PHF1 antibody epitope.

The dose of PHF1 was 250 μg/125 μL dissolved in PBS. Controls wereinjected i.p. with same dose of mouse IgG in PBS. The first injectionwas administered between 9-12 weeks of age. Animals subsequentlyreceived weekly administrations for a total of 13 injections, followedby behavioral testing at 5-6 months and subsequent tissue analysis at6-7 months.

Passive immunization with the PHF1 antibody prevented tau pathologyassociated motor decline in the P301L mouse model. As shown in FIG. 11A,there was a significant difference between IgG injected controls andPHF1 immunized animals on the traverse beam, with control animals havingmore footslips when crossing the beam than immunized animals (trialscombined, p=0.03). Likewise, PHF1 immunized P301L mice had 58% less PHF1stained tau pathology in the dentate gyrus than controls (p=0.02) (FIG.11B). An inverse correlation between plasma levels of PHF1 antibodiesand tau pathology was observed in the brain stem (FIG. 12A; p<0.01), anda strong trend for correlation in the motor cortex (FIG. 12B; p=0.06).

The amount of PHF-1 antibodies (μg/μL) in plasma of immunized animalsdecreased four-fold in two weeks (FIG. 11C). No detectable antibodieswere observed in controls. These are the average values for theimmunized mice.

Example 9 Generation of Monoclonal Tau Antibodies

Ten balb/c mice were immunized with Tau386-408[P-Ser_(396,404)] (SEQ IDNO:13) linked to KLH via a cysteine residue added to the N-terminus.Strong antibody titer was generated against the tau portion of theimmunogen as detected by serial dilutions of plasma (FIG. 13A). Two micewere selected for cell fusion and initial screening was performed withthe immunogen peptide without KLH. Second screening was performed withthe same peptide as well as Tau386-408[P-Ser₃₉₆], Tau386-408[P-Ser₄₀₄]and the non-phospho peptide Tau386-408 (FIG. 13B). Based on thatscreening, clones were selected for the first and second subcloning.Importantly, numerous strongly positive clones were identified (>50) andstable clones have been identified that specifically recognize aphospho-epitope within this region or that bind to a non-phosphorylatedsite within this region, thereby allowing a comparison of the efficacyand safety profile of antibodies binding to a phospho- or non-phosphotau epitopes within the same region of the molecule.

Of the phospho-specific monoclonal antibodies selected for furthersubcloning, four out of six retained their specificity for thephospho-Ser₄₀₄ epitope (see clones 1F12C2, 1F12G6, 4E6E3, and 4E6G7 inFIG. 14A). Two clones are less phospho-specific (8B2D1) or non-specific(8B2D4) (FIG. 14A). Of the non-phospho-specific monoclonal antibodies,6B2E9 and 6B2G12, in particular, retained their non-specificity afterfurther subcloning (FIG. 14B).

The reactivity of the four P-Ser_(396, 404) tau phospho-specific (FIG.15A) and non-phospho-specific (FIG. 15B) monoclonal antibody clones wastested against brain homogenates from the JNPL3 P301L mouse and wildtype(Wt) mouse. Of the four phospho-specific clones, 4E6G7 shows thestrongest reactivity (FIG. 15A), which is consistent with the ELISAresults of FIG. 14A. In contrast with the PHF-1 antibody that alsorecognizes the tau P-Ser_(396, 404) epitope, all clones react betterwith the JNPL3 P301L brain homogenate than the Wt homogenate. Thenon-phospho-specific clones reacted faster, as expected, as most of tauis non-phosphorylated.

Another set of ten balb/c mice was immunized with Tau260-271[P-Ser₂₆₂](SEQ ID NO:12) linked to KLH via a cysteine residue on the C-terminus.Although strong titer was generated against the Tau260-271[P-Ser₂₆₂]immunogen, plasma antibodies recognized the non-phospho peptideTau260-271 as well (FIG. 16A). Eight stable phospho-specific clones wereselected from the second subcloning for further analysis (FIG. 16B) andthe 2C11 clone has been selected for antibody production as it is of theIgG2a isotype. IgG3 has shorter half-life and is therefore notconsidered ideal for passive immunization studies.

The reactivity of the three phospho-specific P-Ser₂₆₂ tau monoclonalantibody clones against brain homogenates from JNPL3 P301L and wildtype(Wt) mice was assessed (FIG. 17). The 2C11 antibody clone recognizes ahigher molecular weight band than the other phospho-specific clones andit does not distinguish between wildtype and P301L tissue. 5F7D10 and5F7E9 are representatives of the other clones. Tau-S recognizes totaltau and binds to an epitope around amino acids 216-227 of tau. CP27recognizes human but not mouse tau.

The 5F7D10 antibody clone readily detected tau pathology in P301L tanglemouse brain sections as shown in FIGS. 18A-18E. The 5F7D10 monoclonalantibody shows strong histological staining in the P301L brain section(FIG. 18A) compared to the wildtype (FIG. 18B). The PHF1 antibody pickedup tau pathology in the same tangle mouse (FIG. 18C) although thepattern was different than with the 5F7D10 antibody, which is notsurprising as they recognize different tau epitopes. FIG. 18D is amagnified image of the boxed region in FIG. 18A depicting neurons withaggregated tau. FIG. 18E is a higher magnified image of tangle-likepathology detected with 5F7D10 in a different JNPL3 P301L mouse.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. A pharmaceutical composition comprising: (A) anantibody, or binding portion thereof, having an antigenic specificityfor an isolated Tau peptide whose amino acid sequence consists of: (a)the amino acid sequence of SEQ ID NO:57; (b) an amino acid sequenceselected from the group consisting of SEQ ID NO:13 and SEQ ID NO:42; (c)an amino acid sequence selected from the group consisting of SEQ IDNO:74 and SEQ ID NO:99; (d) an amino acid sequence selected from thegroup consisting of SEQ ID NO:5 and SEQ ID NO:34; (e) an amino acidsequence selected from the group consisting of SEQ ID NO:23 and SEQ IDNO:52; and (f) an amino acid sequence selected from the group consistingof SEQ ID NO:100-103; and and (B) a pharmaceutically-acceptable carrier,diluent or stabilizer; wherein said composition comprises an amount ofsaid antibody, or binding portion thereof, sufficient to treatAlzheimer's disease or other tauopathy in a recipient subject or toreduce the severity of Alzheimer's disease or said other tauopathy insaid recipient subject.
 2. The pharmaceutical composition of claim 1,wherein said antibody, or binding portion thereof, has an antigenicspecificity for an isolated Tau peptide whose amino acid sequenceconsists of the amino acid sequence of SEQ ID NOs:5, 13, 23 or
 99. 3.The pharmaceutical composition of claim 1, wherein said antibody, orbinding portion thereof, has an antigenic specificity for an isolatedTau peptide whose amino acid sequence consists of the amino acidsequence of SEQ ID NOs:34, 42, 52, 57 or
 74. 4. The pharmaceuticalcomposition of claim 1, wherein said antibody, or binding portionthereof, has an antigenic specificity for an isolated Tau peptide whoseamino acid sequence consists of the amino acid sequence of SEQ IDNOs:100-103.
 5. The pharmaceutical composition of claim 1, wherein saidcomposition additionally comprises one or more additional antibodies, orbinding portions thereof, having an antigenic specificity for a peptidehaving an amino acid sequence selected from the group consisting of SEQID NOs:81-100.
 6. The pharmaceutical composition of claim 4, whereinsaid composition additionally comprises one or more additionalantibodies, or binding portions thereof, having an antigenic specificityfor a peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs:81-100.
 7. The pharmaceutical composition ofclaim 1, wherein said composition additionally comprises one or moreadditional antibodies, or binding portions thereof, having an antigenicspecificity for one or more different amyloidogenic proteins or peptidesselected from the group consisting of an amyloid-beta protein precursor,a prion protein, α-synuclein, amyloid-β, an islet amyloid polypeptide,apolipoprotein AI, apolipoprotein AII, lyzozyme, cystatin C, gelsolin,atrial natriuretic factor, calcitonin, keratoepithelin, lactoferrin, animmunoglobulin light chain, transthyretin, A amyloidosis,β2-microglobulin, an immunoglobulin heavy chain, a fibrinogen alphachain, prolactin, keratin, and medin.
 8. The pharmaceutical compositionof claim 1, wherein said tauopathy is selected from the group consistingof frontotemporal dementia, parkinsonism linked to chromosome 17(FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, progressive subcortical gliosis, tangle only dementia,diffuse neurofibrillary tangles with calcification, argyrophilic graindementia, amyotrophic lateral sclerosis parkinsonism-dementia complex,dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinkerdisease, Hallerworden-Spatz disease, inclusion body myositis,Creutzfeld-Jakob disease, multiple system atrophy, Niemann-Pick diseasetype C, prion protein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, chronic traumatic encephalopathy, andpostencephalitic parkinsonism.
 9. The pharmaceutical composition ofclaim 1, wherein said composition promotes the reduction or clearance ofTau aggregates from the brain of said recipient subject, or reduces theseverity of the formation of Tau aggregates in said recipient subject.10. The pharmaceutical composition of claim 9, wherein said Tauaggregates are neurofibrillary tangles or their pathological Tauprecursors.
 11. The pharmaceutical composition of claim 1, wherein saidcomposition slows progression of Tau pathology-related cognitiveimpairment in said recipient subject, or reduces the severity of saidprogression.
 12. A pharmaceutical composition comprising: (I) anantibody, or binding portion thereof, having an antigenic specificityfor an isolated Tau peptide whose amino acid sequence consists of theamino acid sequence of SEQ ID NO:82; and (II) one or more additionalantibodies, or binding portions thereof, having an antigenic specificityto a peptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs:81-100; and (III) a pharmaceutically-acceptablecarrier, diluent or stabilizer wherein said composition comprises anamount of said antibody, or binding portion thereof, sufficient to treatAlzheimer's disease or other tauopathy in a recipient subject or toreduce the severity of Alzheimer's disease or said other tauopathy insaid recipient subject.
 13. The pharmaceutical composition of claim 12,wherein said composition additionally comprises one or more additionalantibodies, or binding portions thereof, having an antigenic specificityfor one or more different amyloidogenic proteins or peptides selectedfrom the group consisting of an amyloid-beta protein precursor, a prionprotein, α-synuclein, amyloid-β, an islet amyloid polypeptide,apolipoprotein AI, apolipoprotein AII, lyzozyme, cystatin C, gelsolin,atrial natriuretic factor, calcitonin, keratoepithelin, lactoferrin, animmunoglobulin light chain, transthyretin, A amyloidosis,β2-microglobulin, an immunoglobulin heavy chain, a fibrinogen alphachain, prolactin, keratin, and medin.
 14. The pharmaceutical compositionof claim 12, wherein said tauopathy is selected from the groupconsisting of frontotemporal dementia, parkinsonism linked to chromosome17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration,Pick's disease, progressive subcortical gliosis, tangle only dementia,diffuse neurofibrillary tangles with calcification, argyrophilic graindementia, amyotrophic lateral sclerosis parkinsonism-dementia complex,dementia pugilistica, Down syndrome, Gerstmann-Straussler-Scheinkerdisease, Hallerworden-Spatz disease, inclusion body myositis,Creutzfeld-Jakob disease, multiple system atrophy, Niemann-Pick diseasetype C, prion protein cerebral amyloid angiopathy, subacute sclerosingpanencephalitis, myotonic dystrophy, non-guanamian motor neuron diseasewith neurofibrillary tangles, chronic traumatic encephalopathy, andpostencephalitic parkinsonism.
 15. The pharmaceutical composition ofclaim 12, wherein said composition promotes the reduction or clearanceof Tau aggregates from the brain of said recipient subject, or reducesthe severity of the formation of Tau aggregates in said recipientsubject.
 16. The pharmaceutical composition of claim 15, wherein saidTau aggregates are neurofibrillary tangles or their pathological Tauprecursors.
 17. The pharmaceutical composition of claim 12, wherein saidcomposition slows progression of Tau pathology-related cognitiveimpairment in said recipient subject, or reduces the severity of saidprogression.